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  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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  • C12N9/10—Transferases (2.)
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  • C12N2800/00—Nucleic acids vectors
  • C12N2800/10—Plasmid DNA

Definitions

• the present invention relates to the field of circularization of linear double stranded nucleic acids, in particular linear double stranded (ds) DNA nucleic acids.
• ds linear double stranded
• nucleic acid engineering provides many applications, in particular for the purpose of human and veterinary therapy, such as the production of plasmids, notably for the production of recombinant proteins of therapeutic and/or economic interest, creation of DNA libraries for drug screening. These recombinant proteins may be used in turn as drugs to treat diseases.
• polypeptides of therapeutic and/or economic interest encompass, e.g., growth factors, antibodies, hormones, cytokines, enzymes, plasmatic factors, and the like.
• molecular biology For decades, tools have been intensively developed to assemble nucleic acids from various origins, which have been referred to as “molecular biology”.
• One of the main techniques of molecular biology consists in inserting a nucleic acid of interest into a circular nucleic acid vector, which facilitates nucleic acid cloning and amplification.
• human/animal gene encoding a protein of interest may be assembled within bacterial or viral nucleic acid vectors, so as to obtain hybrid circular recombinant nucleic acids enabling target expression of the human/animal protein within the human body.
• This technique relies upon the use of polymerase chain reaction (PCR), restriction enzymes, ligases, and often result in a poor yield, i.e.
• PCR polymerase chain reaction
• circular nucleic acids are one goal to be achieved, because circular nucleic acids are easier to handle, and more resistant to degradation, as compared to single stranded nucleic acids.
• EP2620497 discloses a method for producing a circular DNA from a single DNA molecule and which does not comprise circular DNA formed from multiple-molecule DNA. This method relies upon adding double stranded adapters to one extremity of the nucleic acid to be circularized.
• W02014079900 relates to a method for the in vitro production of supercoiled DNA minicircles having less than 250 base pairs, through a one-pot ligase-mediated circularization reaction of linear nicked double- stranded oligodeoxynucleotide in the presence of a DNA bending protein.
• Chen Cheng et al. J Med Genet 2019; 56:10-17 discloses a method for generating double stranded minicircles vector, which are bacteria-free. This method relies upon amplifying a nucleic acid of interest by PCR using primers introducing restrictions sites at each extremity of said nucleic acid. Digestion of the PCR product with the corresponding restriction enzyme and submitting the result of digestion to ligation allows obtaining circular nucleic acids.
• a first aspect of the invention relates to a method for the circularization of a double stranded DNA nucleic acid, said method comprising the steps of: a) providing a linear or circular double stranded DNA nucleic acid comprising a nucleic acid of the following formula (I):
• - TBC represents a core double stranded DNA nucleic acid to be circularized, in particular having a length of at least about 250 bp, preferably at least about 500 bp, more preferably at least about 1,000 bp;
• HR1 and HR2 represent identical homologous double stranded DNA nucleic acids of identical orientation
• - SORl x and SOR2 y represent nucleic acids having a length of about 5 bp to about 60 bp comprising a site of restriction capable of generating 3’ overhangs having a length of at least 4 nucleotides and having as 3’ terminal nucleotide A, T or G; and x and y are 0 or 1; when x and y are 1, TBC further comprises 2 sites of nicking restriction each located about 0 nucleotide to about 100 nucleotides respectively from the 3’ end of HR1 and from the 3’ end of HR2; - NHRI and NHR2 represent distinct non-homologous double stranded DNA nucleic acids having a length of 0 bp to about 200 bp; bl) when x and y are 0, digesting the circular double stranded DNA nucleic acid comprising a nucleic acid of formula (I) from step a), in the presence of a restriction enzyme that cleaves a restriction site that is
• step a optionally digesting the circular double stranded DNA nucleic acid comprising a nucleic acid of formula (I) from step a), in the presence of a restriction enzyme that cleaves a restriction site that is not located in any one of HR1, HR2 and TBC, so that to generate a linearized nucleic acid;
• step b) or step c) is preceded by, or concomitantly performed with, a step comprising incubating the linear double stranded DNA nucleic acid of step a) with an alkaline phosphatase and/or a polypeptide with a type I exonuclease activity.
• step d) is performed in the presence of divalent and/or trivalent cations.
• step f) is performed in the presence of a polypeptide having a 5’-3’ nuclease activity, preferably Exonuclease VII (Exo VII), RecJ f or Flap endonuclease 1 (FEN1).
• step g) is performed in the presence of a mesophilic DNA ligase, preferably T4 DNA ligase.
• said method further includes a step of binding of the double stranded DNA nucleic acid, in particular the circular double stranded DNA nucleic acid, to one or more non-nucleic acid moiety (moieties), preferably selected in the group comprising linkers, polypeptides, particles, surfaces, and combinations thereof; so as to generate a functionalized binding the double stranded DNA nucleic acid, in particular a functionalized circular double stranded DNA nucleic acid.
• Another aspect of the invention further pertains to a circularized double stranded DNA nucleic acid obtainable by a method according to the instant invention. In some embodiments, said nucleic acid is functionalized and/or relaxed.
• a still other aspect of the invention relates to a host cell comprising a circularized double stranded DNA nucleic acid obtainable by a method according to the instant invention.
• the invention pertains to the circularized double stranded DNA nucleic acid according to the instant invention, for use in gene therapy, and/or in DNA vaccination, and/or in cell therapy, and/or in genome editing, and/or in the production of induced pluripotent stem cells, and/or in the transfection or transformation of cultured cells.
• One aspect of the invention relates to the use of the circularized double stranded DNA nucleic acid according to the instant invention, for the storage of data, and/or for the sequencing of nucleic acids, and/or for the production of rolling circle DNA, and/or for the production of proteins, and/or for the production of RNA, and/or in metabolic pathway engineering, and/or in molecular biology, and/or for the transformation of bacteria, and/or for the production of viruses.
• the invention also relates to a kit for the circularization of a double stranded DNA nucleic acid, said kit comprising: a) a polypeptide having a 3 ’-5’ nuclease activity; b) a polypeptide having a 5’-3’ nuclease activity; c) a DNA polymerase; d) a mesophilic DNA ligase; and optionally, e) one or more buffer(s).
• said kit comprises at least two vials: - the first vial comprising the polypeptide having a 3 ’-5’ nuclease activity; and optionally alkaline phosphatase and/or a polypeptide with a type I exonuclease activity;
• said kit further comprises one or more primer(s) selected in the group consisting of a primer of sequence SEQ ID NO. 43 and a primer of sequence
• “About” preceding a number encompasses plus or minus 10%, or less, of the value of said number. It is to be understood that the value to which the term “about” refers to is itself also specifically, and preferably, disclosed.
• “In vitro” when referred to a method is intended to mean that the steps of the method are performed without the requirement of a living organism, such as, e.g. , a bacterium. In some embodiments, the method may be “substantially performed in vitro”, meaning that most of the steps do not require the presence of a living organism, such as, e.g., a bacterium, and that at least one step of the method may optionally be performed in the presence of said living organism.
• Amplicon is intended to relate to a segment of DNA nucleic acid, e.g., chromosomal DNA nucleic acid, that undergoes amplification, in particular by
• the term “amplicon” relates to a segment of DNA nucleic acid that derives from two or more segments, which can be single stranded or double stranded DNA molecules, intentionally assembled together during the PCR reaction.
• Double stranded nucleic acid is intended to refer to a nucleic acid wherein the two strands, in the 5’-3’ and 3’-5’ orientations, are complementary to one another and hybridize with one another.
• double stranded nucleic acid further refers to a “double stranded nucleic acid molecule”, as a biological entity.
• “Intramolecularly annealed dsDNA nucleic acid” or “product of intramolecular annealing of DNA nucleic acid” refers to the DNA nucleic acid obtained after joining two ends of the same DNA molecule through the hydrogen bonds formed by annealing of the terminal single stranded overlapping overhangs. This “circular” structure of DNA nucleic acids contains 2 gaps, or 2 nicks, or 1 gap and 1 nick, wherein both strains are discontinuous.
• the term “intramolecularly annealed dsDNA nucleic acid” further refers to a “intramolecularly annealed dsDNA nucleic acid molecule”, as a biological entity.
• “Circularized double stranded DNA nucleic acid” refers to a dsDNA nucleic acid that contains one or two continuous strands. As used herein, the term “circularized double stranded DNA nucleic acid” further refers to a “circularized double stranded DNA nucleic acid molecule”, as a biological entity. “Continuous strand” refers to a strand of a nucleic acid that contains no gap and/or no nick. In other words, the nucleotides are linked one to another by a 5 ’ -3 ’ phosphate bond. In other words, each of the nucleotide of the continuous strand is linked to at least one, in particular two, other nucleotide(s). - “Discontinuous strand” refers to a strand of a nucleic acid that contains at least one gap and/or at least one nick.
• Hybridization is intended to refer to the action for two complementary nucleic acid strands, i.e. the strand in the 5’-3’ orientation and the complementary strand in the 3 ’-5’ orientation, to contact in a sense/antisense manner and to form hydrogen bonds.
• Homologous regions is intended to refer to regions displaying similarity of nucleotide sequences. Within the scope of the invention the term “similarity” also encompasses the term “identity”. In practice, homologous regions may hybridize with one another in a sense/antisense manner. Oppositely, “Non-homologous regions” is intended to refer to regions that do not share any sequence similarity or identity.
• nucleic acid identity percentage may be determined using the CLUSTAL W software (version 1.83) the parameters being set as follows: o for slow/accurate alignments: (1) Gap Open Penalty: 15; (2) Gap Extension
• “Recessing” is intended to refer to the action of removing, e.g. by enzymatic digestion, one strand of the extremity of a double stranded nucleic acid. “Overlapping overhangs” is intended to refer to single stranded extremities of nucleic acids that may hybridize with one another.
• “Annealing” or “anneal” is intended to refer to the action of hybridizing 2 complementary nucleic acid strands in a sense/antisense manner.
• Site of restriction is intended to refer to a nucleic acid sequence capable to bind to the corresponding restriction enzyme and to be cleaved in suitable conditions. As used herein, a site of restriction includes the recognition/cleaving site as well as the additional surrounding nucleotide(s) that are required for the enzyme to bind and cleave.
• “Nick” is intended to refer to a discontinuity in a double stranded DNA nucleic acid where there is no phosphodiester bond between adjacent nucleotides of one strand. Within the scope of the instant invention, the terms “nick” and “break” are equivalent and may substitute one another.
• nickase “nicking enzyme” and “nicking (endo)nuclease” are meant to substitute one another and are intended to refer to a polypeptide capable of generating a nick.
• Gap is intended to refer to a single-stranded region within a double-stranded DNA nucleic acid delimited by two double- stranded regions.
• Circularized is intended to refer to the structure of the final product obtainable by the method according to the invention.
• a circularized double-stranded DNA nucleic acid according to the invention refers to a dsDNA nucleic acid that has been circularized following by the method according to the invention.
• “Functionalized nucleic acid” is intended to refer to a nucleic acid that is bound or conjugated to one or more non-nucleic acid moiety (moieties), in particular one or more functional polypeptide(s).
• the functional moiety or polypeptide may facilitate the penetration of nucleic acid molecules into a target recipient cell, facilitate the nuclear localization of the nucleic acid molecules into a target recipient cell, facilitate the localization in cell organelles of the nucleic acid molecules into a target recipient cell, facilitate the visualization of the nucleic acid and/or facilitate the binding to epitopes of choice on cell surface.
• the term “bound” refers to covalent or non-covalent attachment of the moiety or polypeptide to the nucleic acid according to the invention.
• the term “conjugated” is intended to mean that the nucleic acid and the functional moiety or polypeptide are attached one to another by the means of a covalent bond.
• the term “functionalized nucleic acid” further refers to a “functionalized nucleic acid molecule”, as a biological entity.
• “Host cell” is intended to refer to a cell that is the recipient of at least one foreign (or exogenous) nucleic acid molecule.
• Treatment refers to therapeutic treatments wherein the object is to prevent or slow down (lessen) a disorder.
• Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented.
• a subject is successfully "treated” for a disorder if, after receiving a therapeutic amount of a circular double stranded DNA nucleic acid according to the present invention, the subject shows observable and/or measurable reduction in, or absence of, one or more of the following: reduction in the disorder and/or relief to some extent of one or more of the symptoms associated with the disorder; reduced morbidity and mortality, and/or improvement in quality of life issues.
• prevention refers to preventing or avoiding the occurrence of one or more symptom(s) of a disorder.
• the term “prevention” may refer to a secondary prevention, i.e. to the prevention of the re-occurrence of a symptom or a relapse of a disorder. It may also refer, when the disease is cancer, to the prevention of occurrence of metastases after the treatment and/or the removal of a tumor.
• Therapeutic effective amount refers to an amount sufficient to effect beneficial or desired results including clinical results. A therapeutic effective amount can be administered in one or more administrations.
• “Individual” refers to a vertebrate animal, preferably a mammal, more preferably a human. Examples of individuals include humans, non-human primates, dogs, cats, mice, rats, horses, cows, sheep and transgenic species thereof.
• an individual may be a "patient", i.e. a warm-blooded animal, more preferably a human, who/which is awaiting the receipt of, or is receiving medical care or was/is/will be the object of a medical procedure, or is monitored for the development of a disease.
• the individual is an adult (for example a human subject above the age of 18).
• the individual is a child (for example a human subject below the age of 18).
• the individual is a male.
• the individual is a female.
• the inventor showed that it is possible to easily and rapidly circularize double stranded nucleic acids, starting from any double stranded nucleic acid molecule, in particular, any linear or linearized circular double stranded nucleic acid molecule.
• the method of the invention may be performed within a couple of hours through self-hybridization of DNA monomers, and provides one to five orders of magnitude higher yields with up to about 90% efficiency within the range of 100 ng to 10 pg or more, as compared to other methods known in the art. It is worth mentioning that methods known in the art for obtaining circular DNA molecules through hybridization of two or more different DNA monomers, i.e.
• the circularized double stranded DNA nucleic acids obtained by the method of the invention may find many applications, as such as, e.g., cell transfection, gene therapy, storage of information in DNA molecules and for any other use wherein fast and efficient DNA circularization is needed.
• the invention relates to a method for the circularization of a double stranded DNA nucleic acid, said method comprising the steps of: a) providing a linear or circular double stranded DNA nucleic acid comprising a nucleic acid of the following formula (I): NHR 1 - S OR 1 X -HR 1 -TB C -HR2-S OR2 y -NHR2 (I), wherein:
• - TBC represents a core double stranded DNA nucleic acid to be circularized, in particular having a length of at least about 250 bp, preferably at least about 500 bp, more preferably at least about 1,000 bp;
• - HR1 and HR2 represent identical homologous double stranded DNA nucleic acids of identical orientation;
• SORl x and SOR2 y represent nucleic acids having a length of about 5 bp to about 60 bp comprising a site of restriction capable of generating 3’ overhangs having a length of at least 4 nucleotides and having as 3’ terminal nucleotide A, T or G; and x and y are 0 or 1; when x and y are 1, TBC further comprises 2 sites of nicking restriction each located about 0 nucleotide to about 100 nucleotides respectively from the 3’ end of HR1 and from the 3’ end of HR2;
• - NHRI and NHR2 represent distinct non-homologous double stranded DNA nucleic acids having a length of 0 bp to about 200 bp; bl) when x and y are 0, digesting the circular double stranded DNA nucleic acid comprising a nucleic acid of formula (I) from step a), in the presence of a restriction enzyme that cleaves a restriction site that is not located in any one of HR1, HR2 and TBC, so as to obtain a linearized nucleic acid; b2) when x and y are 1, - optionally digesting the circular double stranded DNA nucleic acid comprising a nucleic acid of formula (I) from step a), in the presence of a restriction enzyme that cleaves a restriction site that is not located in any one of HR1, HR2 and TBC, so that to generate a linearized nucleic acid;
• the method is an in vitro method. In certain embodiments, the method is a substantially in vitro method. In some embodiments, the method is performed substantially in vitro. In certain embodiments, the method is performed exclusively in vitro.
• the linear or circular double stranded DNA nucleic acid has a length of at least about 250 bp, preferably at least about 500 bp, more preferably at least about 1,000 bp.
• the expression “at least about 250 bp” encompasses 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1,000, 1,250, 1,500, 1,750, 2,000, 2,250, 2,500, 2,750, 3,000, 3,500, 4,000, 4,500, 5,000, 5,500, 6,000, 6,500, 7,000, 7,500, 8,000, 8,500, 9,000, 9,500, 10,000, 12,000, 14,000, 16,000, 18,000, 20,000, 25,000, 30,000, 35,000, 40, 000, 45,000, 50,000 bp, or more.
• SOR1 and SOR2 represent a nucleic acid having a length of about 5 bp to about 60 bp, preferably of about 7 bp to about 60 bp.
• the term “about 5 bp to about 60 bp” includes 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 and 60 bp.
• the “sites of restriction capable of generating 3 ’ overhangs having a length of at least 4 nucleotides and having as 3’ terminal nucleotide A, T or G” comprised in SOR1 and SOR2 include the recognition/cleaving sites as well as the additional surrounding nucleotide(s) that are required for the enzyme to bind and to efficiently cleave.
• the recognition/cleaving site is surrounded by 1, 2, 3, 4, 5, 6 or more extra nucleotides at its 5’ end and/or its 3’ end, which extra nucleotide(s) participate(s) to the binding of the enzyme of restriction and to the efficient cleaving.
• x and y have the same value, which encompasses 0 and 1. In other words, x and y are 0 and alternatively, x and y are 1.
• TBC comprises 2 sites of nicking restriction each located about 0 nucleotide to about 100 nucleotides respectively from the 3’ end of HR1 and from the
• 0 nucleotide to about 100 nucleotides includes 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
• NHRI and NHR2 represent non-homologous double stranded DNA nucleic acids having a length of 0 bp to about 200 bp.
• 0 bp to about 200 bp includes 0, 1,
• TBC, HR1, HR2 from the linear or circular double stranded nucleic acid of formula (I) are from eukaryotic origin.
• TBC, HR1 and/or HR2 from the linear or circular double stranded nucleic acid of formula (I) are from microbial origin.
• TBC, HR1 and/or HR2 may comprise one or more origin(s) of replication from bacterial origin (e.g . origins of replication pMBl, ColEl or fl) and/or from viral origin (e.g., SV40 origin of replication functional in HEK293T cell line).
• TBC, HR1 and/or HR2 may further comprise one or more nucleic acid(s) encoding one or more polypeptide(s) involved in the resistance to one or more antibiotic(s).
• the TBC, HR1 and/or HR2 may comprise a S/MAR (for scaffold/matrix attachment region) nucleic acid sequence.
• S/MAR nucleic acid sequence also referred to S AR (for scaffold-attachment region) or MAR (for matrix-associated region) are intended to refer to a nucleic acid sequence that is naturally found in the DNA of eukaryotic chromosomes and that promotes attachment to the nuclear matrix.
• the S/MAR nucleic acid sequence may serve as an origin of replication.
• circularized nucleic acid molecules comprising a S/MAR nucleic acid sequence may behave as extra-chromosome in a transfected cell and be advantageously transmitted to its progeny.
• suitable S/MAR nucleic acid sequence may be determined as described in Narwade el al. (Nucleic Acids Research, 2019; 47(14):7247-7261).
• the S/MAR nucleic acid sequence has a length comprised from about 50 bp to about 2,000 bp, preferably from about 400 bp to about 1,500 bp.
• the circularized nucleic acids according to the instant invention may be compatible with a replication step in a eukaryotic cell, and/or in a bacterial cell.
• the circularized nucleic acids according to the instant invention may be compatible with a replication step in cell-free system, such as, e.g., the OriCiro® technology (see https://www.oriciro.com/).
• TBC is a nucleic acid encoding a polypeptide of therapeutic and/or economic interest.
• Non-limitative examples of polypeptide of therapeutic and/or economic interest include antibiotics, anti-fungal compounds, anti-viral compounds, fluorescent polypeptides, growth factors, antibodies (e.g., therapeutic and/or diagnosis antibodies), hormones, cytokines, enzymes, plasmatic factors (e.g., clotting factors), metabolic pathways factors, and the like.
• step a) is performed with an amount of linear or circular double stranded DNA nucleic acid comprising a nucleic acid of formula (I) comprised from about 0.1 ng to about 100 mg.
• the term “about 0.1 ng to about 100 pg” encompasses 0.1 ng, 0.5 ng, 1 ng, 5 ng, 10 ng, 15 ng, 20 ng, 25 ng, 30 ng, 35 ng, 40 ng, 45 ng, 50 ng, 55 ng, 60 ng, 65 ng, 70 ng, 75 ng, 80 ng, 85 ng, 90 ng,
• the linear double stranded DNA nucleic acid of step a) is an amplicon obtained by polymerase chain reaction (PCR).
• the NHRI and NHR2 represent non-homologous double stranded DNA nucleic acids having a length of 1 bp to about 200 bp.
• a DNA nucleic acid of formula (la)-(lo) may be amplified with selected primers in order to achieve the linear double stranded DNA nucleic acid of step a) (see Table 1 below).
• primers suitable to implement the invention also comprise at least 5 nucleotides that hybridize to the starting DNA nucleic acid, as disclosed herein.
• the linear double stranded DNA nucleic acid of step a) is a linear double stranded DNA nucleic acid comprising a nucleic acid of formula (2):
• a DNA nucleic acid of formula (2a), (2b) or (2c) may be amplified with selected primers in order to achieve the linear double stranded DNA nucleic acid of step a) (see Table 2 below).
• the linear double stranded DNA nucleic acid of step a) is a linear double stranded DNA nucleic acid comprising a nucleic acid of formula (3):
• a DNA nucleic acid of formula (3a)-(3h) may be amplified with selected primers in order to achieve the linear double stranded DNA nucleic acid of step a) (see Table 3 below).
• the linear double stranded DNA nucleic acid of step a) is a linear double stranded DNA nucleic acid comprising a nucleic acid of formula (4):
• a DNA nucleic acid of formula (4a)-(4j) may be amplified with selected primers in order to achieve the linear double stranded DNA nucleic acid of step a) (see Table 4 below).
• the linear double stranded DNA nucleic acid of step a) is a linear double stranded DNA nucleic acid comprising a nucleic acid of formula (5):
• a DNA nucleic acid of formula (5a)-(5j) may be amplified with selected primers is order to achieve the linear double stranded DNA nucleic acid of step a) (see Table 5 below).
• the amplicons are raw, i.e., non-purified.
• the amplicons are purified. Methods for purifying amplicons are known in the state of the art (see, e.g., Sambrook el al. (Molecular Cloning- A Laboratory Manual- 2 nd Edition; Cold Spring Harbor Laboratory Press)). Purification may include phenol/chloroform, and/or ethanol precipitation. Purification may also be performed with an appropriate commercial kit, such as, e.g., Monarch® PCR & DNA Cleanup kit from NEB®, StrataPrep® PCR purification kit from Agilent®, following the manufacturer’ s instructions .
• the linear double stranded DNA nucleic acid of step a) may be obtained by amplification and by digestion by the mean of one or more restriction enzymes.
• the circular double stranded DNA nucleic acid comprising a nucleic acid of formula (I) is selected in the group consisting of a chromosome, a plasmid, a cosmid, a bacterial artificial chromosome (BAC), and the like.
• chromosome refers to a genomic DNA nucleic acid molecule, originating from a micro-organism or a eukaryote.
• Plasmid refers to a small extra-genomic DNA nucleic acid molecule, most commonly found as circular double stranded DNA nucleic acid molecules that may for example be used as a cloning vector in molecular biology, to make and/or modify copies of DNA fragments up to about 15 kb ⁇ i.e., 15,000 base pairs). Plasmids may also be used as expression vectors to produce large amounts of proteins of interest encoded by a nucleic acid sequence found in the plasmid downstream of a promoter sequence.
• the term “cosmid” refers to a hybrid plasmid that contains cos sequences from Lambda phage, allowing packaging of the cosmid into a phage head and subsequent infection of bacterial cell wherein the cosmid is cyclized and can replicate as a plasmid.
• Cosmids are typically used as cloning vector for DNA fragments ranging in size from about 32 kb to about 52 kb.
• bacterial artificial chromosome or “BAC” refers to an extra-genomic nucleic acid molecule based on a functional fertility plasmid that allows the even partition of said extra-genomic DNA molecules after division of the bacterial cell.
• BACs are typically used as cloning vector for DNA fragment ranging in size from about 150 kb to about 350 kb.
• the nucleic acid of formula (I) may be a synthetic nucleic acid, i.e., obtained de novo by chemical synthesis, by any suitable method known from the state of the art, or a method adapted therefrom.
• NHRI and NHR2 are distinct non-homologous nucleic acids, they cannot hybridize with each other, with HR1, with HR2 and with any part of TBC in suitable conditions.
• HR1 and HR2 represent identical homologous double stranded DNA nucleic acids of identical orientation, they may hybridize with each other in suitable condition.
• HR1 and HR2 display a AG ranging from about 20 to about 60.
• the term “from about 20 to about 60” include 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,
• measuring the AG parameter may be performed by any suitable method known from the state of the art, or a method adapted therefrom.
• measuring the AG parameter may be performed as disclosed in Wang et al. (Nat Commun 7, 10319 (2016)).
• the linear double stranded DNA nucleic acid of step a) further comprises a 5 ’-phosphate at each 5’ ends.
• NHRI and/or HR1, on one hand, and NHR2 and/or HR2, on the other hand further comprise one or more thiophosphate nucleotide(s).
• thiophosphate nucleotide refers to a nucleotide analogue possessing a thio phosphate bond (PO 3 S) in lieu of a phosphate bond (PO 4 ).
• thiophosphate nucleotides may be commercially available, e.g., from Sigma Aldrich®.
• the linear double stranded DNA nucleic acid of step a) comprises a 5 ’-phosphate at each 5’ ends and one or more thiophosphate nucleotide(s) in NHRI and/or HR1 and in NHR2 and/or HR2 nucleic acid.
• the 5 ’-phosphate and/or the thiophosphate nucleotide(s) is/are provided to the linear double stranded DNA nucleic acid by the mean of PCR amplification.
• the 5 ’-phosphate and/or the thiophosphate nucleotide(s) is/are encompassed within the oligonucleotides.
• the Pfu DNA polymerase is advantageously used to introduce the 5 ’-phosphate and/or the thiophosphate nucleotide(s) into the linear double stranded DNA nucleic acid of step a).
• digesting the circular double stranded DNA nucleic acid comprising a nucleic acid of formula (I) from step a) is performed in the presence of a restriction enzyme that cleaves a restriction site that is not located in any one of HR1, HR2 and TBC, so as to obtain a linearized nucleic acid.
• the site of restriction may be advantageously located within any one of the NHRI or NHR2 nucleic acids, or alternatively between the NHRI and the NHR2 nucleic acids, or anywhere else on the nucleic acid, provided that the restriction site that is not located in any one of nucleic acids HR1, HR2 and TBC.
• enzymatic digestion may be performed by restriction enzymes capable of generating blunt ends, such as, e.g. , Aanl, AccII, Acvl, Afel, Alul, Boxl, BseJI,
• enzymatic digestion may be performed also by restriction enzymes capable of generating 5’-hangouts, such as, e.g., Acc65I, Accl, Acil, AcII, Aflll, Agel, Alwl, ApaLI, ApeKI, Apol, Ascl, Asel, Aval, Avail, AvrII, BamHI, Banl, Bbsl, BbvCI, Bbvl, Bed, BceAI, Bell, BcoDI, Bfal, BfuAI, Bglll, Blpl, BpulOI, BsaHI, Bsal, BsaJI, BsaWI, BseYI, BsiWI, BsmAI, BsmBI, BsmFI, BsoBI, BspDI, BspEI, BspHI, BspMI, BspQI, BsrFal, BsrGI, BssHI, Bst
• enzymatic digestion may be performed by restriction enzymes resulting in 3 ’-hangouts that preferentially carry a 3 '-terminal C nucleotide, such as, e.g., Apal, Banll, Bspl286I, Haell, Kpnl.
• the 3’ hangouts are shorter than or equal to 3 nucleotides, i.e. 1, 2 or 3 nucleotides in length.
• enzymatic digestion may be performed by type IIS restriction enzymes such as, e.g., Aarl, Acc36I, AclWI, Acul, Alw26I, Alwl, AsuHPI, Bbsl, Bbvl, Bccl, BceAI, BciVI, BcoDI, BfuAI, Bful, Bmrl, Bmsl, Bmul, Bpil, Bpml, BpuEI, Bsal, Bse3DI, BseGI, BseMI, BseMII, BseRI.
• type IIS restriction enzymes such as, e.g., Aarl, Acc36I, AclWI, Acul, Alw26I, Alwl, AsuHPI, Bbsl, Bbvl, Bccl, BceAI, BciVI, BcoDI, BfuAI, Bful, Bmrl, Bmsl, Bmul, Bpil, Bpml,
• enzymatic digestion may be performed by type IIS restriction enzymes such as, e.g., Acul, Alwl, Bael, Bbsl, Bbvl, Bccl, BceAI, Bcgl, BciVI, BcoDI, BfuAI, Bmrl, Bpml, BpuEI, Bsal, BsaXI, BseRI, Bsgl, BsmAI, BsmBI, BsmFI, Bsml, BspCNI, BspMI, BspQI, BsrDI, Bsrl, BtgZI, BtsCI, Btsl-v2, BtsIMutI, CspCI, Earl, Ecil, Esp3I, Faul, Fokl, Hgal, Hphl, HpyAV, MboII, Mlyl, Mmel, Mnll, NmeAIII, Plel, Sapl and SfaNI.
• step a optionally digesting the circular double stranded DNA nucleic acid comprising a nucleic acid of formula (I) from step a), in the presence of a restriction enzyme that cleaves a restriction site that is not located in any one of HR1, HR2 and TBC, so that to generate a linearized nucleic acid; - digesting the circular, linear or linearized nucleic acid in the presence of (i) a polypeptide with nickase activity capable of introducing a nick within TBC, and (ii) restriction enzyme(s) capable of digesting the corresponding site of restriction in SOR1 and SOR2.
• the optional digestion of the circular double stranded DNA nucleic acid comprising a nucleic acid of formula (I) from step a) may be performed in the presence of any restriction enzyme capable of generating blunt ends or alternatively resulting in 5’-hangouts or alternatively, less preferentially, resulting in 3’-hangouts.
• step b2) results in the generation of a linear double stranded DNA nucleic acid with 3’ overhangs.
• the restriction enzyme generates 3’ overhangs having a length of at least 4 nucleotides and having as 3’ terminal nucleotide A, T or G. These 3’ overhangs are not prone to digestion by the polypeptide having a 3 ’-5’ nuclease activity, in particular Exo III.
• the polypeptide having a 3 ’-5’ nuclease activity, in particular Exo III would recess the 3’ ends from the nick location.
• the polypeptide with nickase activity is selected in the group consisting of Nt.BspQI, Nt.CviPII, Nt.BstNBI, Nb.BsrDI, Nb.BtsI, Nt.AlwI, Nb.BbvCI, Nt.BbvCI, Nb.BsmI, Nb.BssSI and Nt.BsmAI.
• the polypeptide with nickase activity may be purchased from NEB®.
• about 1 pg to about 5 pg of DNA nucleic acids may be treated with about O.lu to about 5u of a polypeptide with nickase activity.
• treatment with a polypeptide with a nickase activity is or may be performed for about 30 min to about 180 min, preferably for about 60 min to about 90 min.
• treatment with a polypeptide with a nickase activity is or may be performed at a temperature of about 0°C to about 70°C, preferably at a temperature of about 35°C to about 55°C.
• a suitable buffer may be composed of 100 mM NaCl, 50 mM Tris-HCl pH 7.9 at 25°C, 10 mM MgCh, 100 pg/ml BSA (provided by NEB as Buffer lx NEBuffer® 3.1) or composed of 50 mM Potassium Acetate, 20 mM Tris-acetate pH 7.9 at 25°C, 10 mM Magnesium Acetate, 100 pg/ml BSA (provided by NEB as Buffer lx CutSmart® Buffer) or composed for the optimal efficiency of nickase activity.
• lu (1 unit) of Nt.BspQI may be defined as the amount of enzyme required to convert 1 pg of supercoiled pUC19 DNA to open circular form in 1 hour at 50°C in a total reaction volume of 50 pi.
• lu (1 unit) of Nt.CviPII may be defined as the amount of enzyme required to digest 1 pg of pUC19 DNA resulting in a stable pattern of fragments between 25 and 200 bp in 1 hour at 37°C in a total reaction volume of 50 pi.
• lu (1 unit) of Nt.BstNBI may be defined as the amount of enzyme required to digest 1 pg T7 DNA in 1 hour at 55°C in a total reaction volume of 50 pi.
• lu (1 unit) of Nb.BsrDI may be defined as the amount of enzyme required to convert 1 pg of supercoiled pUC19 DNA to open circular form in 1 hour at 65°C in a total reaction volume of 50 pi.
• lu (1 unit) of Nb.BtsI may be defined as the amount of enzyme required to convert 1 pg of supercoiled plasmid FC174 RF I DNA to open circular form in 1 hour at 37°C in a total reaction volume of 50 pi.
• lu (1 unit) of Nt.AlwI may be defined as the amount of enzyme required to convert 1 pg of supercoiled pUClOl DNA (dam-/dcm-) to open circular form in 1 hour at 37°C in a total reaction volume of 50 pi.
• lu (1 unit) of Nb.BbvCI or Nt.BbvCI may be defined as the amount of enzyme required to convert 1 pg of supercoiled plasmid DNA to open circular form in 1 hour at 37°C in a total reaction volume of 50 m ⁇ .
• lu (1 unit) of Nb.BsmI may be defined as the amount of enzyme required to convert 1 pg of supercoiled plasmid pBR322 DNA to open circular form in 1 hour at 65°C in a total reaction volume of 50 pi.
• lu (1 unit) of Nb.BssSI may be defined as the amount of enzyme required to convert 1 pg of supercoiled pUC19 DNA to open circular form in 1 hour at 37°C in a total reaction volume of 50 m ⁇ .
• lu (1 unit) of Nt.BsmAI may be defined as the amount of enzyme required to convert 1 pg of supercoiled plasmid DNA to open circular form in 1 hour at 37°C in a total reaction volume of 50 pi.
• the restriction enzyme capable of generating 3’ overhangs having a length of at least 4 nucleotides and having as 3’ terminal nucleotide A, T or G is selected in the group consisting of Aatll, Ajul, Alol, Arsl, Bael, Bari, Bmtl, Bpll, BspMAI, BspOI, BstNSI, BstXI, EcoT22I, Fael, Fall, Fsel, Gsal, HgiAI, Hinlll, Hpy99I, Hsp92II, I-Ceul, I-Ppol, I-Scel, Mph 11031, Nlalll, Nsil, Nspl, PI-PspI, Pael, Pspl24BI, Psrl, Pstl, Rigl, Sacl, Sbfl, Sdal, Setl, Sphl, Sse8387I, Sstl, Tail, T
• the restriction enzyme capable of generating 3’ overhangs having a length of at least 4 nucleotides and having as 3’ terminal nucleotide A, T or G are those disclosed by Hoheisel (Analytical Biochemistry 1993; 209(2), 238-246).
• the restriction enzyme capable of generating 3’ overhangs having a length of at least 4 nucleotides and having as 3’ terminal nucleotide A, T or G is selected in the group consisting of Ajul, Alol, Arsl, Bael, Bari, Bpll, Fall and Psrl.
• about 1 pg to about 5 pg of DNA nucleic acids may be treated with about O.lu to about lOu of a suitable restriction enzyme capable of generating 3’ overhangs, as defined above.
• treatment with a polypeptide with a restriction enzyme capable of generating 3’ overhangs is or may be performed for about 30 min to about 180 min, preferably for about 60 min to about 120 min.
• treatment with a restriction enzyme capable of generating 3’ overhangs is or may be performed at a temperature of about 0°C to about 45°C, preferably at a temperature of about 20°C to about 40°C.
• step c) comprises recessing both ends of identical orientation of the linear nucleic acid of step a) or the nucleic acid obtained at step bl) or step b2), in the presence of a polypeptide having a 3 ’-5’ nuclease activity, so that HR1 and HR2 are capable of forming overlapping overhangs.
• the term “ends of identical orientation” refers either to the 3’ ends or to the 5’ ends, in particular the 3’ ends on each extremities of the double strand nucleic acid.
• the assessment of a 3 ’-5’ nuclease activity may be performed by standard methods known in the state of the art, or a method adapted therefrom.
• the 3’-5’ nuclease activity may be assessed as disclosed in, e.g., Qiu et al. (J Biol Chem. 2005 Apr 15;280(15):15370-9), or with a commercial dedicated kit, such as, e.g., the BioVision’s 3’ to 5’ Exonuclease Activity Assay Kit from BioVision®, following the manufacturer’s instructions.
• the polypeptide having a 3 ’-5’ nuclease activity is Exonuclease III (also referred to as Exo III).
• Exo III is from E. coli.
• Exo PI may be purchased commercially, e.g., from NEB® or Thermo Fisher Scientific®.
• DNA nucleic acids may be treated with about 0. lu to about lOOu of a polypeptide having a 3’-
• step c) is or may be performed for about 1 min to about 20 min, preferably for about 2 min to about 15 min.
• step c) is or may be performed at a temperature of about 0°C to about 37°C, preferably at a temperature of about 0°C to about 35°C, preferably at a temperature of about 0°C to about 25 °C.
• step c) may be performed on ice or alternatively at a temperature of about 16°C to about 30°C.
• lu (1 unit) may be defined as the amount of the enzyme that catalyzes the release of 1 nmol of acid soluble reaction products from E.coli [ 3 H]-DNA in 30 min at 37 °C; the enzyme activity being assayed in a mixture comprising 50 mM Tris-HCl (pH 8.0), 5 mM MgCh, 1 mM DTT and 0.05 mM sonicated E.coli [ 3 H]-DNA.
• the polypeptide having a 3’-5’ nuclease activity is a DNA polymerase with 3 ’-5’ nuclease activity.
• T4 DNA polymerase is one example of a DNA polymerase with 3 ’-5’ nuclease activity. It is understood that at the end of step c) HR1 and HR2 are capable of forming overlapping overhangs. In other words, HR1 and HR2 form overhangs which are capable of overlapping.
• a column purification kit may provide a mean to remove primers, dNTP and double- stranded primer dimers.
• suitable kits are, e.g., the PCR clean-up NucleoSpin® kits from Macherey-Nagel®.
• an enzymatic PCR cleanup may provide an easy way to remove the remaining primers, dNTP and double- stranded primer dimers left from a PCR reaction.
• step b) or step c) is preceded by, or concomitantly performed with, a step comprising incubating the linear double stranded DNA nucleic acid of step a) with an alkaline phosphatase and/or a polypeptide with a type I exonuclease activity.
• the alkaline phosphatase is the Shrimp Alkaline Phosphatase (SAP), in particular recombinant SAP (rSAP). rSAP is known to dephosphorylate the remaining dNTP.
• SAP Shrimp Alkaline Phosphatase
• rSAP recombinant SAP
• rSAP is known to dephosphorylate the remaining dNTP.
• about 1 pg to about 5 pg of DNA nucleic acids may be treated with about 0.5u to about 5u of alkaline phosphatase.
• treatment with alkaline phosphatase is or may be performed for about 1 min to about 20 min, preferably for about 2 min to about 15 min.
• treatment with alkaline phosphatase is or may be performed at a temperature of about 0°C to about 45 °C, preferably at a temperature of about 30°C to about 40°C, preferably a temperature of about 37°C.
• lu (1 unit) may be defined as the amount of enzyme which catalyzes the hydrolysis of 1 pmol of p-nitrophenyl phosphate per min in glycine buffer (pH 10.4) at 37°C; the enzyme activity being assayed in a mixture comprising 100 mM glycine, pH 10.4, 1 mM MgCh, 1 mM ZnCE, 10 mM p-nitrophenyl phosphate.
• the polypeptide with a type I exonuclease activity is Exonuclease I (also referred to as Exo I).
• Exo I is known to degrade the residual PCR primers.
• Exo I is a thermolabile Exo I.
• the Exo I is from E. coli. In practice, Exo I may be purchased from NEB®.
• about 1 pg to about 5 pg of DNA nucleic acids may be treated with about 0.5u to about 25u of a polypeptide with a type I exonuclease activity.
• treatment with a polypeptide with a type I exonuclease activity is or may be performed for about 1 min to about 20 min, preferably for about 2 min to about 15 min.
• treatment with a polypeptide with a type I exonuclease activity is or may be performed at a temperature of about 0°C to about 45°C, preferably at a temperature of about 30°C to about 40°C, preferably a temperature of about 37°C.
• lu (1 unit) may be defined as the amount of enzyme that will catalyze the release of 2 nmol of acid-soluble nucleotide in a total reaction volume of 100 pi in 6 minutes at 37°C with 0.17 mg/ml single-stranded [ 3 H ⁇ -E.coli DNA; the enzyme activity being assayed in a mixture comprising 100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCh, 100 pg/ml BSA, pH 7.9 at 25°C.
• the hairpins resulting from hybridization of NHRl-SORl x -HRl and HR2-SOR2 y -NHR2, on one hand, or NHRl-SORl x -HRl-TBC and TBC-HR2-SOR2 y -NHR2, on the other hand, upon step b) contain less than 6 nucleotides annealed and are stable at less than about 35°C.
• step d) is or may be performed by first bringing the reaction mixture at a temperature of about 70°C to about 90°C, for about 1 min to about 10 min, preferably for about 5 min, and then by bringing the reaction mixture at a temperature of about 45 °C to about 65 °C, preferably at a temperature of about 50°C to about 60°C, for about 5 min to about 20 min, preferably for about 10 min.
• a temperature of about 70°C to about 90°C allows to deactivate the mesophilic enzymes of the step b) and step c) by thermal treatment and to denature the single stranded ends of double stranded DNA nucleic acid to be circularized in order to release the overlapping overhangs.
• a temperature of about 45°C to about 65°C allows simultaneously to bend the dsDNA molecules with thermodynamic force and to anneal the single stranded overlapping overhangs within the same DNA molecule in order to obtain intramolecularly annealed dsDNA.
• the expression “of about 70°C to about 90°C” encompasses about 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C and 90°C.
• the expression “for about 1 min to about 10 min” encompasses 1 min, 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min and 10 min.
• the expression “of about 45°C to about 65°C” encompasses about 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C and 65°C.
• the expression “for about 5 min to about 20 min” encompasses about 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, 15min, 16 min, 17 min, 18 min, 19 min and 20 min.
• the step d) may be performed by decreasing progressively the temperature of the reaction mixture from a first temperature of about 70°C to about 90°C to a second temperature of about 45 °C to about 65 °C.
• decreasing progressively the temperature is intended to mean that the difference of 2 temperatures is at least 1°C, preferably 2°C and more preferably 5°C.
• decreasing progressively the temperature of the reaction mixture from a first temperature of, e.g., about 85°C to a second temperature of about 45 °C may be performed by decreasing the temperature by 5°C-20°C at a time, i.e., by incubating the reaction mixture as follows: - 85°C for 1 min to 10 min;
• step d) is performed in the presence of divalent and/or trivalent cations.
• the divalent and/or trivalent cations provide annealing conditions favorable for hybridization of the HR1 and HR2 nucleic acids.
• Non-limitative examples of divalent cations include calcium (Ca 2+ ), magnesium (Mg 2+ ) and manganese (Mn 2+ ) cations.
• the divalent cation is magnesium cation.
• the final concentration of the divalent cations, in particular magnesium (Mg 2+ ) in the reaction mixture ranges from about 0.01 mM to about 100 mM, preferably from about 10 to about 50 mM, more preferably from about 25 mM to about 30 mM.
• the divalent cation is magnesium cation.
• the final concentration of the divalent cations, in particular magnesium (Mg2+), in the reaction mixture ranges from about 0.2 mM to about 20 mM.
• the magnesium cations are provided in the form of a magnesium salt, in particular magnesium acetate or magnesium chloride.
• the expression “about 0.01 mM to about 100 mM” includes about 0.01 mM, 0.02 mM, 0.04 mM, 0.06 mM, 0.08 mM, 0.1 mM, 0.2 mM, 0.4 mM, 0.6 mM, 0.8 mM, 1 mM, 1.2 mM, 1.4 mM, 1.6 mM, 1.8 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, 5 mM, 5.5 mM, 6 mM, 6.5 mM, 7 mM, 7.5 mM, 8 mM, 8.5 mM, 9 mM, 9.5 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM
• the final concentration of the divalent cations, in particular magnesium (Mg 2+ ), in the reaction mixture ranges from about 5 mM to about 15 mM, preferably from about 10 mM to about 12 mM.
• Non-limitative examples of trivalent cations include aluminum (Al 3+ ), chromium (Cr 3+ ) and iron III (Fe 3+ ) cations.
• step d) is performed at a pH ranging from about 6.0 to about 10.0, preferentially ranging from about 7.5 to about 8.5, at 25°C.
• the expression “about 6.0 to about 10.0” includes 6.0, 6.1, 6.2, 6.3, 6.4, 6.5,
• step d) is performed in the presence of monovalent cations.
• monovalent cations include silver (Ag + ), copper (Cu + ), lithium (Li + ), potassium (K + ) and sodium (Na + ) cations.
• the final concentration of the monovalent cations in the reaction mixture ranges from about 0 mM to about 150 mM, preferably from about 0 mM to about 100 mM.
• the expression “about 0 mM to about 150 mM” includes 0 mM, 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, 5 mM, 5.5 mM, 6 mM, 6.5 mM, 7 mM, 7.5 mM, 8 mM, 8.5 mM, 9 mM, 9.5 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 10 mM, 11
• the step d) is performed in the presence of one or more additional compound(s), such as, e.g., BSA, DTT, Tween, Tris, and the like.
• the one or more additional compound(s) is/are intended to promote, favorize, annealing of HR1 and HR2 nucleic acids.
• the compounds of the buffer favor efficient enzymatic reactions at appropriate conditions.
• the final concentration in the reaction mixture of the additional compound(s), such as, e.g., BSA, DTT, Tween, Tris, and the like, in particular Tris ranges from about 5 mM to about 150 mM, preferably from about 50 mM to about 100 mM, more preferably from about 70 mM to about 80 mM.
• the expression “about 5 mM to about 150 mM” includes 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 22 mM, 24 mM, 26 mM, 28 mM, 30 mM, 32 mM, 34 mM, 36 mM, 38 mM, 40 mM, 42 mM, 44 mM, 46 mM, 48 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, 105 mM, 110 mM, 115 mM, 120 mM, 125 mM,
• step c) and step d) may be performed concomitantly.
• the term “concomitantly” is intended to mean that the steps are performed in the same vessel.
• the ingredients of the reaction mixture are added in the same vessel and the reaction conditions (temperature and time) of step c) and step d) are as mentioned above.
• step c) and step d) may be followed by treating the reaction mixture at a temperature comprised from about 0°C to about 37°C, preferably a temperature of about 0°C to about 10°C, preferably a temperature of about 2°C to about 25°C, preferably a temperature of about 2°C to about 5°C, for at least 1 min.
• the term “about 0°C to about 37°C” includes 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C,
• the term “at least 1 min” includes 1 min, 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 15 min, 20 min, or longer.
• step d By the end of step d), 2 gaps, 2 nicks, or 1 gap and 1 nick are generated.
• intramolecularly annealed dsDNA i.e., a circular tri-dimensional structure of DNA, is obtained.
• 2 gaps are generated when TBC comprises 2 sites of nicking restriction on different DNA strands each located about 1 nucleotide to about 100 nucleotides respectively from the 3’ end of HR1 and from the 3’ end of HR2.
• 2 nicks are generated when TBC comprises 2 sites of nicking restriction on different DNA strands each located 0 nucleotide respectively from the 3’ end of HR1 and from the 3’ end of HR2.
• step e) is optional when 2 nicks are generated on one strand at step d).
• step e) is performed in the presence of a DNA polymerase with no strand displacement activity and with no 5’ to 3’ exonuclease activity and dNTPs and/or of oligonucleotides with 5 ’-phosphate having a length of about 8 nucleotides to about 100 nucleotides and being complementary to the nucleic acids of the gaps, provided the polypeptide having a 3 ’-5’ nuclease activity has been inactivated.
• step e) is performed in the presence of a DNA polymerase and dNTPs, so as to fill the gaps.
• 2 nicks are generated.
• step e) may be performed in the presence of oligonucleotides and in the presence of a DNA polymerase and dNTPs. In said embodiment, more than 2 nicks are generated.
• the DNA polymerase with no strand displacement activity and with no 5’ to 3’ exonuclease activity is the T4 DNA polymerase.
• T4 DNA polymerase may be purchased from Promega®, NEB®, Takara®, Thermo Fisher Scientific®.
• about 1 pg to about 5 pg of DNA nucleic acids may be treated with about lu to about 50u of a DNA polymerase.
• step e) is or may be performed for about 10 min to about 120 min, preferably for about 30 min to about 90 min.
• step e) is or may be performed at a temperature of about 0°C to about 40°C, preferably at a temperature of about 10°C to about 25°C, preferably at a temperature of about 15°C to about 25°C, preferably a temperature of about 20°C.
• lu (1 unit) of the enzyme catalyzes the incorporation of 10 nmol of deoxyribonucleotides into a polynucleotide fraction in 30 min at 37°C.
• dNTPs include dATP, dCTP, dTTP and dGTP.
• step e) is or may be performed in the presence of about 0.05 mM to about 15 mM of dNTPs, preferably in the presence of about 0.05 mM to about 10 mM of dNTPs, more preferably in the presence of about 1 mM to about 5 mM of dNTPs.
• step e) is performed in the presence of a DNA polymerase and dNTPs, and/or in the presence of oligonucleotides, so as to fill the gaps.
• the length of the gaps would depend of the location of the nicks generated by the polypeptide with nicking activity.
• the oligonucleotides are complementary to the nucleic acids from the gaps.
• the oligonucleotides may have a length of about 8 nucleotides to about 100 nucleotides. In some embodiments, the oligonucleotides have a length of about
• the expression “about 8 nucleotides to about 100 nucleotides” includes 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40,
• step e) is advantageously performed in the presence of a DNA polymerase and dNTPs. In said embodiments, step e) is performed in the absence of oligonucleotides.
• the presence of a polypeptide having 3 ’-5’ endonuclease activity, in particular ExoIII in the reaction mixture may digest oligonucleotides as soon as they anneal to the DNA nucleic acid at room temperature. Therefore, in some embodiments, inactivation of the polypeptide having 3 ’-5’ nuclease activity, in particular ExoIII, may be performed.
• thermal inactivation of the polypeptide having 3 ’-5’ nuclease activity may be performed by thermal inactivation.
• thermal inactivation may comprise heating and cooling of the reaction mixture.
• heating of the reaction mixture may be performed at a temperature ranging from about 70°C to about 90°C, during about 5 min to about 10 min.
• the term “about 70°C to about 90°C” includes 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C and 90°C.
• the term “about 70°C to about 90°C” includes 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C and 90°C.
• the term “about 70°C to about 90°C” includes 70°C, 71°C, 72
• “about 5 min to about 10 min” includes 5, 6, 7, 8, 9 and 10 min.
• step e) may be performed by a competent bacterium.
• the nucleic acid obtained at the end of step d) may be introduced in a competent bacterium, by any suitable method known in the state of the art, or a method adapted therefrom, in particular transformation.
• the method is hence performed substantially in vitro.
• substantially in vitro is intended to mean that at most one step of the method is performed within a living organism, hence in vivo.
• transformation is used herein to refer to the introduction of an extra-genomic nucleic acid in the cytoplasm of a bacterium.
• the bacterium, the cytoplasm of which contains at least one extra-genomic nucleic acid after the step of transformation, are qualified as “transformed bacterium”.
• the transformation of bacteria requires typically that said cells have been beforehand treated to become “competent” for the extra-genomic nucleic acid to enter the cytoplasm upon transformation.
• competent refers to a bacterium that has an increased ability to uptake an extra genomic nucleic acid into its cytoplasm. The skilled artisan is familiar with techniques for preparing competent bacteria.
• the manufacturer for the steps of preparation of competent bacteria, transformation, selection of transformed bacteria one may refer to the manufacturer’s instructions, when commercial kits or materials are used, and/or refer to, e.g., the protocols described by J. Sambrook and D. Russell, Molecular Cloning: A Laboratory Manual, 3 rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. (2001).
• competent bacteria may be chemically competent cells, in particular calcium chloride-treated bacteria.
• competent bacteria may be electrocompetent bacteria.
• chemically competent or electrocompetent bacteria may be purchased from Thermo Fisher Scientific®, Sigma- Aldrich® or NEB®.
• E. coli bacteria a non-limitative list of commercial chemically competent bacteria encompasses BL21(DE3), DH10B, DH5a, Machl, TOP10, INV110, SIG10.
• a non-limitative list of commercial E. coli electrocompetent bacteria encompasses Mega DH10B T1R, ElectroMAX DH5a, One shot TOP 10, SIG10 MAX.
• step f) of removing the NHRI and NHR2 nucleic acids at both 5’ ends, when the length of the NHRI and NHR2 nucleic acids is superior to 0 bp is performed when x and y are 0, i.e. when SOR1 and SOR2 are absent.
• step f) is performed in the presence of a polypeptide having a 5’ -3’ nuclease activity, preferably Exonuclease VII (Exo VII), RecJ f or Flap endonuclease 1 (FEN1).
• the assessment of a 5 ’-3’ nuclease activity may be performed by standard methods known in the state of the art, or a method adapted therefrom.
• the 5 ’-3’ nuclease activity may be assessed as disclosed in, e.g., Lyamichev et al.
• ExoVII, RecJ f or FEN1 nucleases may be purchased from, e.g., NEB®, Thermo Fisher Scientific®.
• step f) is or may be performed for about 30 min to about 120 min, preferably for about 45 min to about 90 min.
• step f) is or may be performed at a temperature of about 0°C to about 37°C, preferably at a temperature of about 15°C to about 25°C, preferably a temperature of about 20°C.
• lu (1 unit) of ExoVII may be defined as the amount of enzyme that will catalyze the release of 1 nmol of acid- soluble nucleotide in a total reaction volume of 50 pi in 30 minutes at 37°C.
• lu (1 unit) is defined as the amount of RecJ f required to produce 0.05 nmol TCA soluble deoxyribonucleotide in a total reaction volume of 50 pi in 30 minutes at 37°C in a suitable buffer 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCh, 1 mM DTT, pH 7.9 at 25°C) with 1.5 pg sonicated single- stranded [ 3 H]-labeled E. coli DNA.
• lu (1 unit) of FEN1 may be defined as the amount of FEN1 required to cleave 10 pmol of 5' DNA flap containing oligonucleotide substrate in a total reaction volume of 10 pi for 10 minutes at 65°C.
• step g) is performed in the presence of a mesophilic DNA ligase, preferably T4 DNA ligase.
• T4 DNA ligase may be purchased from, e.g., Thermo Fisher Scientific®, NEB®, Invitrogen®, Takara®.
• the DNA ligase is a salt resistant DNA ligase.
• the salt resistant DNA ligase may be purchased from NEB® (Salt-T4® DNA ligase).
• step g) is or may be performed for about 15 min to about 120 min, preferably for about 45 min to about 90 min.
• step g) is or may be performed at a temperature of about 0°C to about 37°C, preferably at a temperature of about 15°C to about 25 °C.
• lu (1 unit) catalyzes the exchange of 1 nmol [ 32 P]-labeled pyrophosphate into ATP in 20 min at 37°C; the enzymatic activity being assayed in a mixture comprising 66 mM Tris-HCl (pH 7.6), 6.6 mM MgCk , 10 mM DTT, 66 pM ATP, 3.3 pM [ 32 P] -labeled pyrophosphate.
• step g) is performed in the presence of ATP.
• step g) is or may be performed in the presence of about 0.05 mM to about 15 mM of ATP, preferably in the presence of about 0.1 mM to about 10 mM of ATP, more preferably in the presence of about 1 mM to about 5 mM of ATP.
• the term “about 0.05 mM to about 15.0 mM” includes 0.05 mM, 0.10 mM, 0.15 mM, 0.20 mM, 0.25 mM, 0.30 mM, 0.35 mM, 0.40 mM, 0.45 mM, 0.50 mM, 0.55 mM, 0.60 mM, 0.65 mM, 0.70 mM, 0.75 mM, 0.80 mM, 0.85 mM, 0.90 mM, 0.95 mM, 1.0 mM, 1.5 mM, 2.0 mM, 2.5 mM, 3.0 mM, 3.5 mM, 4.0 mM, 4.5 mM, 5.0 mM, 5.5 mM, 6.0 mM, 6.5 mM, 7.0 mM, 7.5 mM, 8.0 mM, 8.5 mM, 9.0 mM, 9.5 mM, 10.0 mM, 10.5 mM,
• step g) is performed in the absence of any polymer, in particular in the absence of polyethylene glycol (PEG). Said characteristic is advantageous as compared to numerous nucleic acids assembly methods, such as, e.g., those described by US patent n° 8,968,999 (Gibson et al.) as well as those described by Chen Cheng et al. (J Med Genet 2019; 56:10-17), which require the presence of PEG, in particular PEG 4000.
• step e), step f) and step g) may be performed concomitantly.
• circularized double stranded DNA nucleic acids may be assessed with any suitable method known in the state of the art, or a method adapted therefrom.
• the products of a circularization method according to the invention may be analyzed on an agarose gel. Circularized double stranded DNA nucleic acids would appear heavier than the corresponding linear double stranded DNA nucleic acids.
• the method may be performed in vitro , i.e. without the requirement of a living organism, in particular, a bacterium.
• step g) may be performed by a competent bacterium.
• the nucleic acid obtained at the end of step f) may be introduced in a competent bacterium, by any suitable method known in the state of the art, or a method adapted therefrom, in particular transformation.
• step f) when NHRI and NHR2 represent nucleic acids of 0 bp, step f) is not performed, and step e) and step g) may be performed by a competent bacterium.
• the nucleic acid obtained at the end of step d) may be introduced in a competent bacterium, by any suitable method known in the state of the art, or a method adapted therefrom, in particular transformation.
• both strands of the circularized nucleic acid are circular.
• all the nicks generated within the course of the method are sealed.
• said method may further comprise the step of: h) removing the linear and/or non-circularized DNA nucleic acids. In practice this step allows removing both linear and/or non-circularized double-stranded and single- stranded DNA molecules.
• this step may be performed in the presence of a polypeptide having an exonuclease activity, such as, e.g., Exonuclease V (also referred to as Exo V or RecBCD), truncated Exonuclease VIII (also referred to as truncated Exo VIII), T7 exonuclease or Lambda Exonuclease.
• a polypeptide having an exonuclease activity such as, e.g., Exonuclease V (also referred to as Exo V or RecBCD), truncated Exonuclease VIII (also referred to as truncated Exo VIII), T7 exonuclease or Lambda Exonuclease.
• Exo V, truncated Exo VIII or Lambda Exonuclease may be purchased from NEB® or Thermo Fisher Scientific®.
• step h) is or may be performed for about 15 min to about 120 min, preferably for about 45 min to about 90 min.
• step h) is or may be performed at a temperature of about 0°C to about 45°C, preferably at a temperature of about 30°C to about 40°C, preferably at a temperature of about 37°C.
• One unit is defined as the amount of Exo V required to produce 1 nmol of acid- soluble deoxyribonucleotide from double stranded DNA in 30 minutes at 37°C in a total reaction volume of 50 pi; the enzyme activity being assayed in a mixture comprising 1 mM ATP and 50 mM Potassium Acetate, 20 mM Tris-acetate, 10 mM Magnesium Acetate, 1 mM DTT, pH 7.9 at 25°C.
• lu (one unit) is defined as the amount of Lambda Exonuclease required to produce 10 nmol of acid-soluble deoxyribonucleotide from double- stranded substrate in a total reaction volume of 50 pi in 30 minutes at 37°C in the suitable buffer (67 mM Glycine-KOH, 2.5 mM MgCh, 50 pg/ml BSA, pH 9.4 at 25°C), with 1 pg sonicated duplex [ 3 H]-DNA.
• suitable buffer 67 mM Glycine-KOH, 2.5 mM MgCh, 50 pg/ml BSA, pH 9.4 at 25°C
• step h) may be followed by treating the reaction mixture with EDTA, preferably at a final concentration ranging from about 1 mM to about 25 mM, more preferably at a final concentration ranging from about 5 mM to about 15 mM, and/or at a temperature ranging from about 60°C to about 80°C, for 15 min to 60 min.
• EDTA preferably at a final concentration ranging from about 1 mM to about 25 mM, more preferably at a final concentration ranging from about 5 mM to about 15 mM, and/or at a temperature ranging from about 60°C to about 80°C, for 15 min to 60 min.
• the term “about 1 mM to about 25 mM” includes 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM and 25 mM.
• the method further includes a step of binding the double stranded DNA nucleic acid, in particular the circularized double stranded DNA nucleic acid, to one or more non-nucleic acid moiety (moieties), preferably selected in the group comprising linkers, polypeptides, particles, surfaces, and combinations thereof; so as to generate a functionalized binding of the double stranded DNA nucleic acid, in particular a functionalized circularized double stranded DNA nucleic acid.
• the term “binding” encompasses both covalent and non-covalent binding. In practice, covalent binding may also refer to as “conjugation”.
• this step is a functionalization step.
• functionalization may be performed at various stages of the method.
• linkers, polypeptides may be introduced during PCR amplification, so as to obtain a functionalized linear nucleic acid of formula (I).
• functionalization may be performed by the means of one or more modified primer(s) capable of introducing said linker and/or said polypeptide into the HR1 and/or HR2 or TBC.
• functionalization may also be performed during step d), by the mean of modified dNTP nucleotides, or by the mean of modified oligonucleotides.
• functionalization may be performed as a late stage of the method, i.e., onto a circularized double stranded DNA nucleic acid according to the invention.
• step e) further includes annealing of one or two or several oligonucleotides, preferentially modified with one to ten polypeptides and/or one to ten linkers, with the intramolecularly annealed dsDNA molecule providing one or two gaps.
• the site or the sites for the oligonucleotide annealing are localized within gap region of TBC.
• the length and the sequence of the oligonucleotide assures specific annealing, as it is well known in the state of the art, and may range from 8 nucleotides to 100 nucleotides, preferentially from 18 nucleotides to 30 nucleotides.
• the said functional polypeptides are well known in the state of the art, and are employed to facilitate the penetration of nucleic acid molecules into a target recipient cell, to facilitate the nuclear localization of the nucleic acid molecules into a target recipient cell, to facilitate the localization in cell organelles of the nucleic acid molecules into a target recipient cell, and/or to facilitate the binding to epitopes of choice on cell surface.
• the polypeptide facilitating the penetration into a target recipient cell may comprise, or consist of, a cell-penetrating peptide (CPP), also referred to as “protein transduction domain” (PTD).
• CPP cell-penetrating peptide
• PTD protein transduction domain
• cell-penetrating peptides refer to peptides being generally short in length, i.e., of up to 30 residues, having a net positive charge and acting in a receptor-independent and energy-independent manner.
• the functional polypeptide according to the invention may comprise one or more CPPs.
• the CPP may advantageously be cleavable upon penetration into a recipient cell.
• Examples of CPPs include those selected in the group consisting of hydrophilic CPPs, hydrophobic CPPs, and amphipathic CPPs.
• hydrophilic CPPs are peptides composed mainly by hydrophilic amino acids usually rich in amino acid residues R and K
• amphipathic CPPs are peptides that contain an alternating pattern of polar/charged amino acids and non-polar, hydrophobic amino acids, and are usually rich in amino acid residue K.
• hydrophobic CPPs are peptides containing only apolar residues, with low net charge or have hydrophobic amino acid groups that are crucial for cellular uptake.
• hydrophilic CPPs may be selected in the non-limitative group comprising Antennapedia Penetratin (RQIKWFQNRRMKWKK, SEQ ID NO. 1), BMV Gag-(7-25) (KMTR AQRRA A ARRNRWT AR, SEQ ID NO. 2), D-Tat
• GRKKRRQRRRPPQ SEQ ID NO. 3
• FHV Coat-(35-49) RRRRNRTRRNRRRVR, SEQ ID NO. 4
• HTFV-II Rex-(4-16) TRRQRTRRARRNR, SEQ ID NO. 5
• PTD-4 PTDRRKKFRRFK, SEQ ID NO. 6
• PTD-5 RRQRRT S KFMKR, SEQ ID NO. 7
• R9-Tat GRRRRRRRRRPPQ, SEQ ID NO. 8
• SynBl RGRFSYSRRRFSTSTGR, SEQ ID NO. 9
• SynB3 RRFSYSRRRF, SEQ ID NO. 10
• TAT Y GRKKRRQRRR, SEQ ID NO.
• amphipathic CPPs may be selected in the non-limitative group comprising Transportan (GWTLNSAGYLLGKINLKALAALAKKIL, SEQ ID NO. 12), MAP (KLALKLALKLALALKLA, SEQ ID NO. 13), SBP (MGLGLHLLVLAAALQGAW S QPKKKRKV, SEQ ID NO. 14),
• FBP GALFLGWLG AAGSTMGAW S QPKKKRKV, SEQ ID NO. 15
• MPG GALFLGFLG AAGSTMGAW S QPKKKRKV, SEQ ID NO. 16
• MPCJ (ANLS) (GALFLGFLGAAGSTMGAWSQPKSKRKV, SEQ ID NO. 17), Pep-1 (KETWWETWWTEW S QPKKKRKV, SEQ ID NO. 18), and
• the CPP may be comprised in a sequence selected in a group comprising SEQ ID NO. 1 to SEQ ID NO. 19.
• the CPPs may be such as those disclosed in the patent applications WO 2011/157713 and WO 2011/157715 (Hoffmann La Roche®), or derivatives thereof.
• the polypeptide facilitating the penetration into a target recipient cell may comprise, or consist of, a nuclear localization domain.
• suitable classical or non-classical nuclear localization domains may be such as those disclosed in, e.g., Lange et al. (2007), Kosugi et al. (2009) and Marfori et al. (2011).
• the nuclear localization domain may be selected in the non-limitative group comprising the sequences PAAKRVKLD (SEQ ID NO. 20) of c-Myc, MS RRRKANPTKLS EN AKKLAKE VEN (SEQ ID NO. 21) of EGL-13, PAATKKAGQA (SEQ ID NO. 22) or KRPAATKKAGQAKKKK (SEQ ID NO. 23) of nucleoplasmin, and PKKKRKV (SEQ ID NO. 24) of SV40.
• PAAKRVKLD SEQ ID NO. 20
• MS RRRKANPTKLS EN AKKLAKE VEN SEQ ID NO. 21
• PAATKKAGQA SEQ ID NO. 22
• KRPAATKKAGQAKKKKKK SEQ ID NO. 23
• PKKKRKV SEQ ID NO. 24
• the nuclear localization domain may be comprised in a sequence selected in a group comprising SEQ ID NO. 20 to SEQ ID NO. 24.
• the polypeptide facilitating the penetration into a target recipient cell may comprise, or consist of, an organelle localization domain.
• organelle is intended to refer to a specific cellular compartment, such as, e.g., the chloroplast, the endoplasmic reticulum (ER), the mitochondria, the peroxisome, the vacuoles, and the like.
• SEQ ID NO. 25 may promote ER localization; the sequence MLS LRQS IRFFKP ATRTLCS S R YLL (SEQ ID NO. 26) may promote mitochondria localization; the sequence MV AM AM AS LQS SMSSLSLSSNS FLGQPLS PITLS PFLQG (SEQ ID NO. 27) may promote chloroplast localization; the sequences SKL and XXXXRLXXXXHL (SEQ ID NO. 28), wherein X may represent any amino acid residue, may promote peroxisome localization; the sequences HSRFNPIRLPTTHEPA (SEQ ID NO. 29), S S S S FADS NPIRP VTDR A AS TLE (SEQ ID NO. 30), VFAE AIA AN S TLV AE (SEQ ID NO. 31), NGLLVDTM (SEQ ID NO. 32), V S GG VWDS S VETN AT AS LV S EM (SEQ ID NO. 33) and
• QAHPNFPLEMPGSDEVAK may promote vacuole localization.
• the organelle localization domain may be comprised in a sequence selected in a group comprising SEQ ID NO. 25 to SEQ ID NO. 34.
• the double stranded DNA nucleic acid according to the invention may be covalently bound to the at least one functional polypeptide by the mean of a linker.
• linker is intended to refer to an organic entity that covalently or non-covalently attaches the functional polypeptide to the double stranded nucleic acid of the invention.
• the linker may comprise from 1 to 1,000 plural valent atoms selected from the group consisting of C, N, O, S and P.
• the linker may be linear or non-linear; and some linkers may have pendant side chains or pendant functional groups (or both).
• the linker may be a Gly-rich linker, in particular comprising one or more “GS” motifs, such as, e.g., GGSSG (SEQ ID NO. 35), GSGSGS (SEQ ID NO. 36), GGSGGSGGSGG (SEQ ID NO. 37), GGGGSLVPRGSGGGGS (SEQ ID NO. 38), GGSGGHMGSGG (SEQ ID NO. 39), SGGGSSHS (SEQ ID NO. 40), SGGSGGSSHS (SEQ ID NO. 41), and SGGSGGSGGSSHS (SEQ ID NO. 42).
• the linker may be biotin or streptavidin.
• the functional polypeptide may be preferentially bound to an oligonucleotide in order to purify it prior annealing and, next, the polypeptide- oligonucleotide conjugate may be annealed to the gap region of intramolecularly annealed dsDNA and sealed at the step g) with a mesophilic ligase.
• the double stranded DNA nucleic acid according to the invention may be bound to, in particular, covalently bound to, one or more antibody/ies, fragment(s) thereof, afucosylated antibody/ies, diabody/ies, triabody/ies, tetrabody/ies, nanobody/ies, and analog(s) thereof.
• an “antibody”, also referred to as immunoglobulins (abbreviated “Ig”), is intended to refer to a gamma globulin protein that is found in blood or other bodily fluids of vertebrates, and is used by the immune system to identify and neutralize foreign objects, such as bacteria and viruses.
• Antibodies consist of two pairs of polypeptide chains, called heavy chains and light chains that are arranged in a Y-shape. The two tips of the Y are the regions that bind to antigens and deactivate them.
• antibody as used herein includes monoclonal antibodies, polyclonal antibodies, multi-specific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological activity.
• immunoglobulin immunoglobulin
• an “antibody fragment” comprises a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody.
• antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870; Zapata et ah, Protein Eng. 8(10): 1057-1062 [1995]); single-chain antibody molecules; and multi- specific antibodies formed from antibody fragments.
• One may refer to a “functional fragment or analog” of an antibody, which is a compound having qualitative biological activity in common with a full-length antibody.
• a functional fragment or analog of an anti-IgE antibody is one that can bind to an IgE immunoglobulin in such a manner so as to prevent or substantially reduce the ability of such molecule from having the ability to bind to the high affinity receptor, Fc[epsilon]RI.
• Papain digestion of antibodies produces two identical antigen binding fragments, called “Fab” fragments, and a residual “Fc” fragment, a designation reflecting the ability to crystallize readily.
• the Fab fragment consists of an entire F chain along with the variable region domain of the H chain (VH), and the first constant domain of one heavy chain (CHI). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site.
• F(ab')2 antibody fragments differ from Fab fragments by having additional few residues at the carboxy terminus of the CHI domain including one or more cysteines from the antibody hinge region.
• Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group.
• F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments that have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
• an “afucosylated antibody” refers to an antibody lacking core fucosylation. As a matter of fact, nearly all IgG-type antibodies are N-glycosylated in their Fc moiety. Typically, a fucose residue is attached to the first N-acetylglucosamine of these complex-type N-glycans. In other words, an “afucosylated antibody” refers to an antibody that does not possess N-glycans.
• the term “diabody” refers to a small antibody fragment prepared by constructing sFv fragments (see preceding paragraph) with short linkers (about 5-10 residues) between the VH and VF domains such that inter-chain but not intra- chain pairing of the V domains is achieved, resulting in a bivalent fragment, i.e. , fragment having two antigen-binding sites.
• Bispecific diabodies are heterodimers of two “crossover” sFv fragments in which the VH and VF domains of the two antibodies are present on different polypeptide chains.
• Diabodies are described in more details in, e.g., EP 404,097; WO 93/11161; and Hollinger et al, Proc. Natl.
• a “triabody” is intended to refer to an antibody that has three Fv heads, each consisting of a VH domain from one polypeptide paired with the VL domain from a neighboring polypeptide.
• a “nanobody” refers to a functional antibody that consists of heavy chains only and therefore lack light chains. These heavy-chain only antibodies contain a single variable domain (VHH) and two constant domains (CH2, CH3).
• VHH variable domain
• CH3 constant domain
• antibodies may be bound to the circularized DNA at the last step of the method according to the instant invention.
• the particles are nanoparticles, microparticles, or a combination thereof.
• Circularized double stranded nucleic acids according to the invention may be bound to a surface, such as e.g., a vessel, a petri dish, a fabric, or a cellular surface.
• the invention relates to a method, in particular an in vitro method, for the circularization and the functionalization of a double stranded DNA nucleic acid.
• the method comprises the steps as defined herein.
• x and y are 0, and the linear double stranded DNA nucleic acid comprises a nucleic acid of the formula (II): NHR1-HR1-TBC-HR2-NHR2 (II).
• the method for the circularization of a double stranded DNA nucleic acid comprises the steps of: a) providing a linear or circular double stranded DNA nucleic acid comprising a nucleic acid of the following formula (II):
• NHR 1 -HR 1 -TBC-HR2-NHR2 (II), wherein: - TBC represents a core double stranded DNA nucleic acid to be circularized, in particular having a length of at least about 250 bp, preferably at least about 500 bp, more preferably at least about 1,000 bp;
• HR1 and HR2 represent identical homologous double stranded DNA nucleic acids of identical orientation
• - NHRI and NHR2 represent distinct non-homologous double stranded DNA nucleic acids having a length of 0 bp to about 200 bp; b) digesting the circular double stranded DNA nucleic acid comprising a nucleic acid of formula (II) from step a), in the presence of a restriction enzyme that cleaves a restriction site that is not located in any one of HR1, HR2 and TBC, so as to obtain a linearized nucleic acid; c) recessing both ends of identical orientation of the linearized nucleic acid obtained in step b), in the presence of a polypeptide having a 3’-5’ nuclease activity; d) annealing the DNA nucleic acid obtained at step c), thereby generating 2 gaps; e) filling the 2 gaps; f) optionally removing the NHRI and/or NHR2 at one or both 5’ ends, when the length of the NHRI and NHR2 is superior to 0 bp
• the method for the circularization of a double stranded DNA nucleic acid comprises the steps of: a) providing a linear or circular double stranded DNA nucleic acid comprising a nucleic acid of the following formula (III):
• TBC is a core double stranded DNA nucleic acid to be circularized, in particular having a length of at least about 250 bp, preferably at least about 500 bp, more preferably at least about 1,000 bp; TBC further comprises 2 sites of nicking restriction each located about 0 nucleotide to about 100 nucleotides respectively from the 3’ end of HR1 and from the 3’ end of HR2;
• HR1 and HR2 represent identical homologous double stranded DNA nucleic acids of identical orientation
• SOR1 and SOR2 represent nucleic acids having a length of about 5 bp to about 60 bp comprising a site of restriction capable of generating 3’ overhangs having a length of at least 4 nucleotides and having as 3’ terminal nucleotide A, T or G;
• - NHRI and NHR2 represent distinct non-homologous double stranded DNA nucleic acids having a length of 0 bp to about 200 bp; bl) optionally digesting the circular double stranded DNA nucleic acid comprising a nucleic acid of formula (III) from step a), in the presence of a restriction enzyme that cleaves a restriction site that is not located in any one of HR1, HR2 and TBC, so that to generate a linearized nucleic acid; b2) digesting the circular, linear or linearized nucleic acid in the presence of (i) a polypeptide with nickase activity capable of introducing a nick within TBC, and (ii) restriction enzyme(s) capable of digesting the corresponding site of restriction in SOR1 and SOR2; c) recessing both ends of identical orientation of the nucleic acid of step b2) in the presence of a polypeptide having a 3 ’-5’ nuclease activity; d)
• the circularization may be performed in one single vessel, in the presence of at least one inhibitor of the Pfu DNA polymerase; a polypeptide having a 3 ’-5’ nuclease activity, in particular ExoIII; polypeptides having an exonuclease activity, in particular Exol, and Lambda exonuclease; a DNA polymerase, in particular Hot-Start Taq DNA polymerase; a DNA ligase, in particular Taq ligase; dNTPS; ATP; and appropriate buffers.
• the Lambda exonuclease recesses 5’ ends up to the first thiophosphate nucleotide it encounters. In other words, the Lambda exonuclease recesses 5’ ends but cannot recess thiophosphate bonds. In practice, exonuclease Exol recesses 3’ ends up to a 5’-phosphate end, so as to provide blunt ends.
• the inhibitor of the Pfu DNA polymerase may be any one of the compounds described in Sun et al. (Sci Rep 2018; 8:1990) or in Lasken et al. (J Biol Chem 1996; 271(30): 17692- 17696).
• the 3’ ends of the linear double stranded nucleic acid of step a) are or may be recessed in the presence of ExoIII and Exol, whereas the 5’ ends of the linear double stranded nucleic acid of step a) are recessed in the presence of Lambda exonuclease.
• the circularized double stranded DNA nucleic acid according to the invention comprises the one or more thiophosphate nucleotide(s), as defined above.
• the S atom of the thiophosphate nucleotide(s) allows site-specific functionalization of the circularized double stranded DNA nucleic acid according to the invention.
• the circularized double stranded DNA nucleic acid according to the invention is exclusively from bacterial and/or viral origin.
• the term “exclusively from bacterial and/or viral origin” is intended to mean that at least 75% of the circularized double stranded DNA nucleic acid according to the invention is from bacterial and/or viral origin.
• the term “at least 75%” encompasses 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100%.
• the term “exclusively from bacterial and/or viral origin” is intended to mean that at least 75% of the circularized double stranded DNA nucleic acid according to the invention is from bacterial and/or viral origin.
• the term “at least 75%” encompasses 75%, 76%, 77%, 78%, 79%, 80%,
• “from bacterial and/or viral origin” is intended to mean that the circularized double stranded DNA nucleic acid comprises bacterial and/or viral DNA.
• the circularized double stranded DNA nucleic acid according to the invention is free of bacterial and/or viral nucleic acid.
• the circularized double stranded DNA nucleic acid as defined herein is at least 75% from human origin, preferably at least 80%, preferably at least 90%, more preferably at least 95% from human origin.
• the circularized double stranded DNA nucleic acid according to the invention is 100% from human origin.
• the above-described methods may be performed in less than about 3h.
• the method as defined herein may be performed in about lh30 to about 3h00.
• the term “about lh30 to about 3h00” encompasses, about lh30, lh35, lh40, lh45, lh50, lh55, 2h00, 2h05, 2hl0, 2hl5, 2h20, 2h25, 2h30, 2h35,
• the yield of the method is at least about 25%, preferably at least about 50%, preferably at least about 75%, more preferably at least about 90%.
• yield is intended to refer to the amount of circularized nucleic acid molecules over the amount of total nucleic acid molecules (both linear and circular nucleic acid molecules).
• the expression “at least about 25%” includes at least about 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%,
• the yield of circularization is about 95%.
• Another aspect of the invention relates to a circularized double stranded DNA nucleic acid obtainable by a method according to the instant invention.
• the circularized double stranded DNA nucleic acid is functionalized and/or relaxed.
• the circularized double stranded DNA nucleic acid is a functionalized DNA nucleic acid. In certain embodiments, the circularized double stranded DNA nucleic acid is a relaxed DNA nucleic acid. In certain embodiments, the circularized double stranded DNA nucleic acid is a functionalized and relaxed DNA nucleic acid.
• the circularized double stranded DNA nucleic acid is a supercoiled DNA nucleic acid.
• the topology of circularized DNA produced with the use of the instant invention may be further modified with proteins well known in the state of the art, for example such as topoisomerases of types I, II, III, IV, V and VI.
• the host cell according to the invention may be a eukaryotic cell or a prokaryotic cell.
• eukaryotic host cells may include animal cells, fungi cells, plant cells or yeast cells.
• the host cell according to the invention may be an animal cell, including e.g., a non-human mammal cell and a human cell.
• an animal host cell may be selected in a non-limitative group comprising a cell of the central nervous system, an embryonic cell, an endothelial cell, an epithelial cell, a germ cell, a hematopoietic progenitor cell, a hematopoietic stem cell, an induced Pluripotent Stem Cell (iPSC), a muscular cell, a progenitor cell, a stem cell, and the like.
• iPSC induced Pluripotent Stem Cell
• animal ESCs in particular human ESCs may advantageously be obtained without embryo destruction, as described by Chung et al. (2008), or by parthenogenetic activation of an unfertilized oocyte, as described by Sagi et al. (2016).
• the host cell may belong to a tissue selected in a non-limitative group comprising a connective tissue, an endothelial tissue, an epithelial tissue, a muscle tissue, a nervous tissue, a vascular tissue, and the like.
• the host cell may belong to an organ selected in a non-limitative group comprising a bladder, a bone, a brain, a breast, a central nervous system, a cervix, a colon, an endometrium, a kidney, a larynx, a liver, a lung, an esophagus, an ovarian, a pancreas, a pleura, a prostate, a rectum, a retina, a salivary gland, a skin, a small intestine, a soft tissue, a stomach, a testis, a thyroid, an uterus, a vagina.
• a bladder a bone, a brain, a breast, a central nervous system, a cervix, a colon, an endometrium, a kidney, a larynx, a liver, a lung, an esophagus, an ovarian, a pancreas, a pleura, a prostate, a
• the host cell may non-limitatively originate from a human or a non-human animal, in particular a dog, a cat, a mouse, a rat, a fly, a rabbit, a pig, a chicken, a mosquito, a zebrafish, a horse and a cow.
• the host cell may originate from a vegetal in particular, bean, com, rice, soy, tomato, and wheat.
• the host cell may be a microorganism, in particular selected in a group comprising archaea, bacteria and protozoa.
• suitable bacteria may be exclusively non-pathogenic bacteria.
• non-pathogenic refers to a bacterium that does not harm or cause an infection in a target vegetal or animal, in particular a mammal animal, more preferably a human.
• suitable bacteria may be an attenuated pathogenic bacterium.
• the expression “attenuated pathogenic” refers to a bacterium that has a reduced virulence, as compared to a non-attenuated pathogen.
• Non-limitative examples of attenuated pathogenic bacteria may comprise attenuated bacteria of the genus Pseudomonas, preferably P. aeruginosa ⁇ , of the genus Listeria, preferably L. monocytogenes.
• the bacterium is a Gram-negative bacterium. In practice, the Gram coloration may be performed according to the methods well described in the state of the art.
• the bacterium is from the family of Enterobacteriaceae.
• said bacterium is of the genus Escherichia, preferably of the species E. coli.
• the bacterium is a Gram-positive bacterium.
• Gram-positive bacteria include bacteria of the genus Bifidobacterium, and lactic acid bacteria (LAB), in particular of the genus Abiotrophia, Aerococcus, Carnobacterium, Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Oenococcus, Pediococcus, Streptococcus, Tetragenococcus, Vagococcus and Weissella.
• the host cell may be a genetically modified cell.
• Circularized double stranded DNA nucleic acids according to the invention may be incorporated in the host cell by any suitable method known in the state of the art, or a method adapted therefrom.
• bacterial host cells may be transformed, whereas the eukaryotic host cells may be transfected with the circularized double stranded DNA nucleic acids according to the invention.
• Chemical transformation or transfection methods may encompass the use of calcium chloride. Mechanical transformation or transfection methods may include electroporation.
• Sambrook et al. Molecular Cloning- A Laboratory Manual- 2 nd Edition; Cold Spring Harbor Laboratory Press.
• the circularized double stranded DNA nucleic acid according to the instant invention is for use as a medicament.
• the invention further relates to the use of a circularized double stranded DNA nucleic acid according to the instant invention for the preparation or the manufacture of a medicament.
• a still other aspect of the invention relates to the circularized double stranded DNA nucleic acid according to the invention for use in gene therapy, and/or in DNA vaccination, and/or in cell therapy, and/or in genome editing, and/or in the production of induced pluripotent stem cells, and/or in the transfection or transformation of cultured cells.
• Another aspect of the invention relates to the use of the circularized double stranded DNA nucleic acid according to the invention for gene therapy, and/or for DNA vaccination, and/or for cell therapy, and/or for genome editing, and/or for the production of induced pluripotent stem cells, and/or for the transfection or transformation of cultured cells.
• a further aspect of the invention relates to a method for implementing gene therapy, and/or DNA vaccination, and/or cell therapy, and/or genome editing, and/or production of induced pluripotent stem cells, and/or transfection or transformation of cultured cells, comprising at least step a) to step g), as defined herein.
• the expression “gene therapy” is intended to refer to a therapeutic technique that relies upon genes to treat or prevent a disease. In practice, this technique consists in introducing a gene into a patient’s cells, in order to either replace a mutated gene that causes disease with a healthy copy of the gene, inactivate (or “knock out”) a mutated gene that is functioning improperly, or introduce a new gene into the body to help fight a disease.
• DNA vaccination is intended to refer to the direct introduction into appropriate tissues of a DNA vector containing the DNA sequence encoding the antigen(s) against which an immune response is sought, and relies on the in-situ production of the target antigen (World Health Organization).
• the expression “cell therapy” is intended to refer to a therapeutic technique that relies upon cells to treat or prevent a disease. In practice, this technique consists in transplanting cells into a diseased tissue or organ in order to restore the tissue or organ function.
• the cell therapy includes therapy mediated by adoptive T cell therapy, in particular chimeric antigen receptor (CAR) T-cells.
• adoptive T cell therapy includes culturing and expanding tumor infiltrating lymphocytes (TIL), isolating and expanding one particular lymphocyte T cell clone, as well as using T cells that have been engineered to express a chimeric antigen receptor (CAR) to potently recognize tumor antigens and destroy tumor cells.
• TIL tumor infiltrating lymphocytes
• CAR chimeric antigen receptor
• the circularized DNA nucleic acids according to the invention encode a chimeric antigen receptor (CAR).
• gene editing is intended to refer to the targeted insertion, deletion, modification or substitution of nucleic acids within the recipient’s genome.
• the invention also relates to the therapeutic use of a circularized double stranded DNA nucleic acid according to the invention for preventing and/or treating a disorder.
• the invention further relates to a method for preventing and/or treating a disorder in an individual in need thereof, comprising the administration of a therapeutic effective amount of a circularized double stranded DNA nucleic acid according to the invention.
• the disorder may be selected in a non-limitative group comprising a cancer, a genetic disorder, an infectious disease and a neurodegenerative disease.
• the cancer is selected in a non-limitative group comprising a bladder cancer, a bone cancer, a brain cancer, a breast cancer, a central nervous system cancer, a cervix cancer, a cancer of the upper aero digestive tract, a colorectal cancer, an endometrial cancer, a germ cell cancer, a glioblastoma, a Hodgkin lymphoma, a kidney cancer, a laryngeal cancer, a leukemia, a liver cancer, a lung cancer, a myeloma, a nephroblastoma (Wilms tumor), a neuroblastoma, a non-Hodgkin lymphoma, an esophageal cancer, an osteosarcoma, an ovarian cancer, a pancreatic cancer, a pleural cancer, a prostate cancer, a retinoblastoma, a skin cancer, a small intestine cancer, a soft tissue sar
• the genetic disorder may be selected in the non-limitative group comprising Achondroplasia, Alpha- 1 Antitrypsin Deficiency, Antiphospholipid Syndrome, Autism, Autosomal Dominant Polycystic Kidney Disease, Breast cancer, Charcot-Marie-Tooth, Colon cancer, Cri du chat, Crohn's Disease, Cystic fibrosis, Dercum Disease, Down Syndrome, Duane Syndrome, Duchenne Muscular Dystrophy, Fanconi Anemia, Factor V Leiden Thrombophilia, Familial Hypercholesterolemia, Familial Mediterranean Fever, Fragile X Syndrome, Gaucher Disease, Hemochromatosis,
• Hartnup's Disease Hemophilia, Holoprosencephaly, Huntington's disease, Kartagener's Syndrome, Klinefelter syndrome, Marfan syndrome, Myotonic Dystrophy, Neurofibromatosis, Noonan Syndrome, Osteogenesis Imperfecta, Parkinson's disease, Phenylketonuria, Tru Anomaly, Porphyria, Progeria, Prostate Cancer, Retinitis Pigmentosa, Severe Combined Immunodeficiency (SCID), Sickle cell disease, Skin Cancer, Spinal Muscular Atrophy, Tay-Sachs, Thalassemia, Trimethylaminuria, Tuberous Sclerosis, Turner Syndrome, Velocardiofacial Syndrome, WAGR Syndrome and Wilson Disease.
• SCID Severe Combined Immunodeficiency
• the infectious disease may be selected in the non-limitative group comprising Acute rheumatic fever, Anthrax, Australian bat lyssavirus, Avian influenza (Bird Flu), Babesiosis, Barmah Forest virus, Botulism, Brucellosis, Campylobacteriosis, Chancroid, Chickenpox, Chikungunya, Chlamydia, Cholera, Creutzfeldt- Jakob disease (CJD), Cryptosporidiosis, Cytomegalovirus (CMV), Dengue, Dientamoeba fragilis, Diphtheria, Donovanosis, Ebola virus disease, Epidemic keratoconjunctivitis, Epstein-Barr virus (EBV), Fifth disease, Gastroenteritis, German measle (Rubella), Giardiasis, Gonorrhoea, Glandular fever (Infectious mononucleosis), Haemolytic uraemic
• Coli (STEC/VTEC), Shigellosis, Shingles, Smallpox, Syphilis, Tetanus (lock-jaw), Tuberculosis (TB), Tularemia, Typhoid, Typhus, Varicella-Zoster virus, Viral haemorrhagic fevers, Whooping cough, Yellow fever and Zika virus.
• the neurodegenerative disease may be selected in the non-limitative group comprising Alzheimer's disease, Amyotrophic lateral sclerosis, Down’s syndrome, Friedreich's ataxia, Huntington's disease, Lewy body disease, Parkinson's disease and Spinal muscular atrophy.
• the invention relates to the use of the circularized double stranded DNA nucleic acid according to the instant invention, for the storage of data, and/or for the sequencing of nucleic acids, and/or for the production of rolling circle DNA, and/or for the production of proteins, and/or for the production of RNA, and/or in metabolic pathway engineering, and/or in molecular biology, and/or for the transformation of bacteria, and/or for the production of viruses.
• a further aspect of the invention relates to a method for implementing the storage of data, and/or the sequencing of nucleic acids, and/or the production of rolling circle DNA, and/or the production of polypeptides, and/or the production of RNA, and/or metabolic pathway engineering, and/or molecular biology, and/or the transformation of bacteria, and/or for production of viruses comprising at least step a) to step g), as defined herein.
• the invention also relates to a method for implementing the storage of data, and/or the sequencing of nucleic acids, and/or the production of rolling circle DNA, and/or the production of polypeptides, and/or the production of RNA, and/or metabolic pathway engineering, and/or molecular biology, and/or the transformation of bacteria, and/or for production of viruses comprising at least a step of implementing a circularized nucleic acid obtained by a method according to the invention.
• the circularized nucleic acid according to the invention may encode one or more polypeptide(s), or one or more RNA(s).
• the circularized nucleic acid according to the invention may be suitable for the production of polypeptides, or for the production of RNA.
• Transfection of eukaryotic cells or transformation of bacteria may be performed with the circularized nucleic acid according to the invention, by the mean of any suitable method known from the state of the art, or a method adapted therefrom.
• the circularized nucleic acid according to the invention may encode one or more polypeptide involved in the metabolic pathway.
• the circularized nucleic acid according to the invention may be useful to reconstitute a metabolic pathway into the host cell.
• the circularized nucleic acid according to the invention may be used as a vector in order to clone nucleic acids of interest in microorganisms or to propagate the vector through replication in eukaryotic cells.
• the circularized nucleic acid according to the invention may provide a tool for molecular biology.
• the circularized nucleic acid according to the invention may encode the genetic material for producing viruses.
• digital data may be stored in the form of the circularized nucleic acid according to the invention.
• the NHRI or NHR2 nucleic acids may serve as index, i.e. may represent information suitable for easily retrieving the digital data from the vial containing a set of different digital data stored in the form of circularized DNA molecules.
• the digital data may be deciphered with the use of a DNA sequencer machine, preferentially based on those technologies that require DNA molecules providing the features of the rolling circle DNA molecules, preferentially the technology of Pacific Biosciences®.
• the circularized nucleic acids obtained with the use of the instant invention may be applied for all those applications when the rolling circle DNA molecules are needed.
• the invention relates to a kit for the circularization of a double stranded DNA nucleic acid, said kit comprising: a) a polypeptide having a 3 ’-5’ nuclease activity; b) a polypeptide having a 5 ’-3’ nuclease activity; c) a DNA polymerase; d) a mesophilic DNA ligase; and optionally, e) one or more buffer(s).
• the polypeptide having a 5 ’-3’ nuclease activity, the DNA polymerase and the DNA ligase are in the form of a mixture.
• said kit further comprises one or more buffer(s).
• the kit comprises a buffer comprising ATP and dNTPs.
• the kit comprises at least two vials:
• the first vial comprising the polypeptide having a 3 ’-5’ nuclease activity; and optionally alkaline phosphatase and/or a polypeptide with a type I exonuclease activity;
• the second vial comprising a DNA ligase; and optionally a polypeptide having a
• nuclease activity 5 ’-3’ nuclease activity, and/or a DNA polymerase with no strand displacement activity and with no 5’ to 3’ exonuclease activity, ATP and dNTPs.
• the kit comprises at least two vials:
• the first vial comprising the polypeptide having a 3 ’-5’ nuclease activity
• the second vial comprising a DNA ligase.
• the first vial further comprises alkaline phosphatase and/or a polypeptide with a type I exonuclease activity.
• the second vial further comprises a polypeptide having a 5’-3’ nuclease activity.
• the second vial further comprises a DNA polymerase with no strand displacement activity and with no 5’ to 3’ exonuclease activity, and dNTPs.
• the polypeptide having a 3 ’-5’ nuclease activity and optionally alkaline phosphatase and/or the polypeptide with a type I exonuclease activity within the first vial are in an appropriate buffer.
• DNA ligase, and optionally the polypeptide having a 5 ’-3’ nuclease activity, the DNA polymerase with no strand displacement activity and with no 5’ to 3’ exonuclease activity and dNTPs within the second vial are in an appropriate buffer.
• kits further comprises one or more primers and/or oligonucleotides with functional modifications.
• the kit further comprises one or more primer(s) selected in the group consisting of a primer of sequence SEQ ID NO. 43 (TTAGATTAGTTCATGGTCATAGCTGTTTCCTGTTTTCCCAGTCACGACG) and a primer of sequence SEQ ID NO. 44 (CCT G AGCGG AT A AC A ATTT C AC AC ) .
• the above-described method allows producing circularized nucleic acid molecules: - in a fast, cheap and simple manner, as compared to existing methods;
• - that may behave as an extra-chromosome, when comprising a S/MAR nucleic acid sequence
• - may be compatible with existing technologies such as, e.g., lentivirus, sleeping beauty, CRISPR/Cas9, “DNA minicircles”, and replicative episome;
• Figure 1 is a scheme illustrating one embodiment of the invention.
• the starting dsDNA nucleic acid comprises TBC, HR2, NHR2.
• an amplicon of formula NHR1-HR1-TBC-HR2-NHR2 is obtained.
• Said amplicon is incubated with Exo III nuclease in order to generate 5 ’-overhangs.
• An annealing step generate a circular DNA nucleic acid comprising 2 gaps and 2 non-hybridized 5’-overhangs.
• the 2 gaps are then filled in the presence of a DNA polymerase and the 5 ’-overhangs are further removed in the presence of nuclease with 5 ’-3’ activity (Exo 5 ’-3’), hereby generating a circular dsDNA comprising 2 nicks. These 2 nicks are finally sealed in the presence of a ligase.
• the final product is circularized dsDNA nucleic acid, in which the 2 strands are continuous.
• FIG. 2 is a scheme illustrating one embodiment of the invention.
• the starting dsDNA nucleic acid is plasmid comprising a nucleic acid of formula NHR 1 -S OR 1 -HR 1 -TBC-HR2-S OR2-NHR2.
• the starting dsDNA nucleic acid is digested in the presence of restriction enzymes cleaving the corresponding restriction sites on SOR1 and SOR2, and in the presence of nicking restriction enzymes, that cleave the nick restriction sites on each strand of TBC.
• One nick restriction site is localized at distance of the 3 ’ end of HR1
• the other nick restriction site is localized at 0 nucleotide of the 3’ end of HR2.
• the resulting nucleic acid comprises 2 nicks and 2 3’-overhangs, which cannot be digested by the Exo III nuclease.
• Digestion by Exo III nuclease generates a dsDNA nucleic acid with 3’-overhangs, since digestion initiated at the nick restriction sites.
• Annealing generates a circular dsDNA nucleic acid with 1 gap and 1 nick.
• the gap is filled by the mean of an oligonucleotide, which results in a dsDNA nucleic acid having 3 nicks, which are sealed in the presence of a ligase.
• Figure 3 is a scheme illustrating one embodiment of the invention.
• the starting dsDNA nucleic acid comprises TBC, HR2, NHR2’, wherein NHR2’ represent partial NHR2.
• an amplicon of formula NHR1-HR1-TBC-HR2-NHR2 is obtained.
• NHRI further comprises a restriction site.
• Said amplicon is incubated with the restriction enzyme that cleaves the restriction site within NHRI, which generates a dsDNA nucleic acid of formula HR1-TBC-HR2-NHR2, in which the HR1 presents a 5 ’-overhang.
• the resulting dsDNA nucleic acid is digested in the presence of an Exo III nuclease in order to generate 5’-overhangs.
• An annealing step generates a circular dsDNA nucleic acid comprising 2 gaps and 1 non-hybridized 5’-overhangs, formed of NHR2.
• the 2 gaps are filled in the presence of a DNA polymerase, generating 2 nicks.
• the 5’-overhangs is not removed, and a ligase seals only one of the 2 nicks, generating a circular dsDNA nucleic acid, in which only one of two strands is continuous, also named “rolling circle DNA”.
• the NHR2 5 ’-overhang may be useful as an index, e.g., when the rolling circle DNA is intended to be employed for data storage.
• Figure 4 is a photograph showing the efficacy of intramolecular annealing of dsDNA with respect to the annealing temperature.
• Lane L ladder (the 1.5 kbp, 5.0 kbp and 20.5 kbp nucleic acids are represented on the left side); lane 1: 80°C; lane 2: 75°C; lane 3: 70°C; lane 4: 65°C; lane 5: 60°C; lane 6: 55°C; lane 7: 50°C.
• Arrows indicate the nature of the nucleic acid; a: intramolecularly annealed dsDNA; b: trimers; c: dimers; d: linear DNA (3.8 kbp).
• Figure 5 is a photograph showing that the treatment of non-purified PCR product with Exo III, rSAP and Exo I improves the output of intramolecular annealing of dsDNA.
• Lane L ladder; lane K: control, amplicons DNA before treatments and without purification (linear; 3.8 kbp);
• lane A 12 min treatment on ice with mix of 5u Exo I+lu rSAP+25u Exo III in 50 pL of final 0.5xMgAcetate containing 5 pL of PCR product;
• lane B 3 min treatment on ice with mix of 5u Exol+lu rSAP+25u Exo III in 50 pL of final 0.5xMgAcetate containing 5 pL of PCR product;
• lane 1 only 5 u Exo I treatment at 37°C for 20 min and 25 u Exo III on ice for 3 min or 10 min;
• lane 2 lu rSAP treatment at 37°C during 20 min
• FIG. 1 is a photograph showing the stability of intramolecularly annealed dsDNA with respect to the ratio DNA/Exo III.
• Lane L ladder; lane K: control, amplicons DNA before treatments; lane 1: 100 ng/10 pL; lane 2: 300 ng/10 pL; lane 3: 700 ng/10 pL; lane 4: 900 ng/10 pL.
• Amounts of Exo III are indicated in the upper part of the photograph (lOu, 20u and 40u). Arrows indicate the nature of the nucleic acid; a: intramolecularly annealed dsDNA; b: dimers; c: linear DNA (3.8 kbp).
• Figure 7 is a photograph showing the efficiency of intramolecular annealing of dsDNA in the presence of Exo I.
• Lane L ladder; lane 1: lOu Exo III; lane 2: 40u Exo III. Arrows indicate the nature of the nucleic acid; a: intramolecularly annealed dimeric dsDNA; b: intramolecularly annealed monomeric dsDNA; c: linear DNA (3.8 kbp).
• Figure 8 is a photograph showing the efficiency of intramolecular annealing of dsDNA with respect to the amount of T4 DNA Polymerase. Lane 1: purified amplicon 1.8 kbp
• Figure 9 is a photograph showing the efficiency of circularization with respect to the ratio between DNA ligase, Exo VII and T4 DNA Polymerase.
• the buffer contains 2 mM ATP and 2 mM dNTP.
• Lane A Amplified fragment before circularization; lane B: After Exo
• lane 1 40u DNA Ligase, lu Exo VII and 5u T4 Pol
• lane 2 40u DNA Ligase, O.lu Exo VII and 5u T4 Pol
• lane 3 40u DNA Ligase, O.Olu Exo VII and 5u T4 Pol
• lane 4 40u DNA Ligase, lu Exo VII and 0.5u T4 Pol
• lane 5 40u DNA Ligase, O.lu Exo VII and 0.5u T4 Pol
• lane 6 40u DNA Ligase, O.Olu Exo VII and 0.5u T4 Pol
• lanes 1-6 300 ng of DNA in 20 mE. Arrows indicate the nature of the nucleic acid; a: intramolecularly annealed dsDNA;
• Figure 10 is showing the efficiency of circularization with respect to the ratio between DNA ligase, Exo VII and T4 DNA Polymerase.
• the buffer contains 2 mM ATP and 2 mM dNTP.
• Lane A Amplified fragment before circularization
• lane B After Exo III lOOu for 5 min at RT° of 3000 ng of DNA in 100 pL 0.5xMgAcetate buffer and anneal 88°C for 5min, 50°C for 15min
• lane 1 200u DNA Ligase, 5u Exo VII and 2.5u T4 Pol
• lane 2 lOOu DNA Ligase, 2.5u Exo VII and 1.25u T4 Pol
• lane 3 50u DNA Ligase, 1.25u Exo VII and 0.6u T4 Pol
• lane 4 25u DNA Ligase, 0.6u Exo VII and 0.3u T4 Pol
• lane 5 12u DNA Ligase, 0.3u Exo VII and 0.15u T4 Pol
• Figure 11 is a scheme illustrating one embodiment of the invention. 5’ phosphates ends and thiophosphate nucleotide analogues may be introduced in the HR1 and HR2 and circularization may be obtained via a two-step process.
• Figure 12 is a scheme illustrating one embodiment of the invention in the presence of thiophosphate nucleotide analogues in the linear double stranded nucleic acid to be circularized.
• Figure 13 is a scheme illustrating circularization process of Figure 13, in which the step of recessing the ends of the linear double stranded nucleic acid to be circularized by Lambda exonuclease, Exol et ExoIII are further detailed.
• Figure 14 is a photograph showing the efficiency of DNA molecules circularization as being depend on magnesium concentration. The DNA molecules were incubated with Exonuclease III in a buffer containing different magnesium acetate (MgAc) concentration (from 0 mM to 26 mM) for 2 min at 30°C, 10 min at 75°C, 5 min at 60°C and 1 min 4°C, successively.
• MgAc magnesium acetate
• Lane 1 molecular weight marker (see corresponding indication on a side in bp); lane 2: 0 mM MgAc; lane 3: 2 mM MgAc; lane 4: 6 mM MgAc; lane 5: 10 mM MgAc; lane 6: 14 mM MgAc; lane 7: 18 mM MgAc; lane 8: 22 mM MgAc; lane 9: 26 mM MgAc; lane 10: molecular weight marker is relaxed circular DNA of a plasmid 3868 bp obtained by treatment with a nickase Nt.BbvCI; lane 11: molecular weight marker (in bp).
• nucleic acid a: circular 4,528 bp nucleic acid; b: concatemers of the 4,528 bp fragments; c: linear 4,528 bp fragment; d: linear 1,901 bp (from the plasmid backbone).
• Figure 15 is a photograph showing that the method according to the invention provides high yields of circularized DNA molecules at high concentration of DNA molecules.
• the DNA molecules were incubated with Exonuclease III in 30 mM magnesium acetate buffer for 2 min at 30°C, 10 min at 75°C, 5 min at 60°C and 1 min at 4°C, successively.
• 30 ng/pL of DNA molecules in 12 pL reaction volume were treated with 20u of Exonuclease III (lane 3) and 150 ng/pL DNA molecules in 50 pL were treated with lOOu of Exonuclease III (lane 4).
• Lane 1 molecular weight markers (bp; see corresponding indication on a side); lane 2: relaxed circular DNA of a plasmid 3,868 bp obtained by treatment with a nickase Nt.BbvCI; lane 3: DNA molecules treated at 30 ng/pL concentration; lane 4: DNA molecules treated at 150 ng/pL concentration; lane 5: molecular weight marker. Arrows indicate the nature of the nucleic acid; a: circularized 4,528 bp DNA molecule; b: linear 1,901 bp fragment (from the plasmid backbone).
• Figure 16 is a set of photographs showing that the DNA molecules produced with the circularization method according to the invention promote an increased efficiency of cell transfection. Fig.
• FIG. 16A Human osteosarcoma cells (HOS ATCC® CRL-1543TM) transfected with circular 4,528 bp DNA molecules (black bars) display higher percentage of GFP-positive cells compared to parental 6,711 bp plasmid DNA (white bars) at all tested transfection conditions. The transfection conditions were 1,000 ng, 750 ng, 500 ng or 250 ng of DNA molecules applied for transfection per well in 24-well plate.
• Fig. 16B Lung carcinoma cells of A549 cell line (ATCC® CCL-185TM) transfected with circular 4,528 bp DNA molecules (black bars) display higher percentage of GFP-positive cells compared to parental 6,711 bp plasmid DNA (white bars) in all tested transfection conditions.
• EXAMPLES Human osteosarcoma cells
• Example 1 embodiments according to the invention
• Figures 1, 2 and 3 illustrate three embodiments for obtaining a circularized dsDNA nucleic acid according to the invention (see the corresponding legends).
• Example 2 protocol for circularizing a DNA nucleic acid
• This product may be directly applied for transfection of cells in culture using a kit for transfection such as Lipofectamine® or for electroporation of cells in culture or for transformation of competent bacteria using an electroporation or thermal shock.
• the buffer 0.5xMgAcetate Buffer (work dilution) is as follows:
• This buffer is to be added at the step 1 of the protocol.
• This buffer preferentially contains ATP (final concentration in reaction 1 mM - 5 mM) and dNTP (final concentration in reaction 1 mM - 5 mM) if optional treatment with rSAP was void at the first step of the protocol.
• a modified Mg/ Acetate Buffer may be added at the step 3) of the protocol (Magnesium Acetate concentration may be at 0-1 mM and Potassium Acetate may be at 0-50 mM).
• This buffer preferentially contains ATP (final concentration in reaction 1 mM - 5 mM) and dNTP (final concentration in reaction 1 mM - 5 mM) if ATP and dNTP were absent in the 0.5X Mg/Acetate Buffer to be added at the step 1 of the protocol.
• Figure 4 shows that intramolecularly annealed dsDNA (see arrow a) can be obtained at every annealing temperature tested. However, lower temperatures (60°C, 55°C and 50°C) result in a higher efficacy of intramolecular annealing (see the intensity of the intramolecularly annealed dsDNA (arrow a), as compared to the other forms (linear (arrow d); dimer (arrow c) and trimer (arrow b)).
• Example 4 Exol and rSAP treatment improved intramolecular annealing starting from raw, non-purified PCR product a) Experimental design The experiments have been performed as follows: 5 pL of raw PCR product was diluted to 50 pL of 0.5xMgAcetate buffer. Enzymes Exo I and rSAP were added or not and incubated if necessary, at 37°C during 20 min. There was added 25u of Exo III, incubated for 3 min or 10 min or 12 min and annealed using thermal treatment 88°C for 5min, 50°C for 3min, 4°C for lmin. b) Results ( Figure 5)
• the rSAP dephosphorylates dNTP nucleotides so that the polymerase from PCR reaction does not repair the degraded 3’-DNA strands.
• Exo I eliminates unused primers so that they do not compete with homologous sequences during circularization by annealing.
• the treatments by only Exo I or only rSAP during 20 min at 37°C prior Exo III treatment and annealing display slight improvement or no improvement of intramolecular annealing as compared to the non-treated control DNA (see intense lower bands and relatively weak upper bands on Lanes 1, 2 and 4 in Figure 5).
• Example 5 Efficiency of intramolecular annealing with respect to amounts of amplicon and Exo III a) Experimental design 100 ng to 900 ng of purified amplicons (3.8kbp) were treated with different concentrations of Exo III (lOu, 20u, 40u) in 10 pL of reaction volume and incubated during 5 minutes on ice. Annealing was performed at 88°C for 5min, 50°C for 3min, 3°C for 1 min. b) Results ( Figure 6) Figure 6 shows that the treatments at high Exo III concentrations lead to significant degradation of intramolecularly annealed dsDNA (compare the Lanes 1 to 4 after 10 u, 20 u and 40 u of Exo III concentration (arrow a)).
• T4 DNA polymerase has less potential “to open” intramolecularly annealed dsDNA molecules and “to monomerize” linear concatemers if applied at the temperature lower than 36°C. DNA monomers appear after elongation with T4 DNA polymerase at 36°C (see low intense band on Lane le in Figure 8). Optimal temperature for T4 polymerase- dependent elongation is at 25°C or lower (compare the major band on Lane 1 to the major lower band on Lanes la, lb, lc and Id in Figure 8). The phenomena of “DNA monomerisation” derives from quasi- strand-displacement activity (that is absent in T4 polymerase) at relatively high temperature.
• DNA-wobbling “displaces” the complementary strand in front of T4 DNA polymerase, which progressively elongates the 3 ’-end through “displaced” strand until “opens” the circle of intramolecularly annealed dsDNA.
• Example 8 Efficiency of the DNA circularization with respect to the reparation of gaps a) Experimental design
• the intramolecularly annealed dsDNA molecules were first treated with different ratios (see below) of three enzymes in mixtures containing DNA Ligase, Exonuclease VII and T4 polymerase in 0.5xMgAcetate buffer with 1.25 mM dNTP, 0.5 mM ATP and 300 ng of DNA in 20 pL of reaction volume during 1 hour at room temperature ( Figure 9).
• the results depicted in Figure 10 show that: - the concentration of one or more than one of enzymes in the mix is above optimal in conditions of 1 and 2 that leads to transformation of intramolecularly annealed dsDNA and dimeric DNA into linear monomers (see intense lower band in 1 and 2 and relatively weak bands in la and 2a); the concentration of enzymes is near to optimal in conditions of 3 and 4 because the production of circularized DNA is at maximum after Exo V/RecBCD treatment (see intense band in 3a and 4a) and because the production of linear monomers is moderate (see intense upper band and presence of dimers in 3 and 4); the concentration of enzymes is lower than optimal in conditions of 5 to 8 because the intensity of circularized DNA band gradually decreases from 5 to 8 after Exo V/RecBCD treatment (see the band intensity in 5a, 6a, 7a and 8a).
• Example 9 Example of a two-step circularization protocol ( Figures 11-13)
• Figure 11 shows that it is possible to introduce thiophosphate nucleotide analogs (asterisks), as well as 5’ phosphate ends (Phos 5’), in the HR1 and HR2 nucleic acids by the means of appropriate oligonucleotides (primers).
• a linear double stranded nucleic acid is obtained, which can be circularized, without the purification step, in a one step process in the presence of an inhibitor of Pfu DNA polymerase (InhibOfPfu-Pol is an oligonucleotide containing uracil nucleotides), Exonuclease III (ExoIII), Exonuclease (Exol), Exonuclease Lamdba (ExoLambda), Hot Start Taq DNA polymerase (Hot-Start-TaqPol), Taq DNA ligase (TaqLigase), dNTPS, ATP and the appropriate buffer.
• an inhibitor of Pfu DNA polymerase is an oligonucleotide containing uracil nucleotides
• Exonuclease III ExoIII
• Exonuclease Exol
• Exonuclease Lamdba ExoLambda
• the 3’ ends are recessed in the presence of ExoIII and Exol, and the 5’ ends are recessed in the presence of Lambda exonuclease.
• the presence of thiophosphate nucleotide analogs does not interfere in the recessing of 3’ ends by ExoIII.
• Lambda exonuclease recesses 5’ ends up to the thiophosphate nucleotide analog, where it stops recessing.
• Exol recesses 3’ ends until reaching the 5’ phosphate on the other strand and stops recessing, so as to form blunt ends of each ends of the linear nucleic acid.
• ExoIII recesses the 3’ ends.
• a linear DNA for circularization is obtained by enzymatic cleavage of a cloned plasmid.
• the plasmid (6,711 bp) is cut at one Stul site and two BsrBI sites providing three fragments: 4,528 bp of a sequence to be circularized, 1,901 bp and 282 bp of bacterial backbone.
• the fragment 4,528 bp obtained by cleavage at Stul and BsrBI sites contains the sequence CCTGTGTGAAATTGTTATCCG (SEQ ID NO: 45) repeated on its both 5’ terminus and 3’ terminus.
• the plasmid was treated with Stul and BsrBI restriction enzymes in CutSmart® buffer from New England Biolabs® following standard protocol.
• the purified DNA (final concentration 30 ng/pL in 10 pL) was mixed with a buffer containing Tris- Acetate pH 8.073 mM, Potassium Acetate 60 mM, different Magnesium Acetate concentrations from 0 mM to 26 mM.
• 20u of Exonuclease III in 2 pL the same buffer was added and incubated as following: 30°C for 2 min, 75°C for 10 min, 60°C for 5 min, 4°C for 10 min, successively.
• the product of the reaction was loaded on an agarose gel for electrophoresis.
• the circularized relaxed DNA have very similar localization as compared to the molecular weight marker (lane 10) that corresponds to the relaxed plasmid DNA 3,868 bp obtained by treatment with the nicking enzyme Nt.BbvCI.
• Example 11 Efficiency of the circularization method at high concentration of DNA molecules a) Experimental design
• the plasmid was treated with Stul (Mbil) and BsrBI (Ecol47I) restriction enzymes in Anza® buffer from Thermo Fisher Scientific® following standard protocol.
• the purified DNA molecules (final concentration 30 ng/pL in 10 pL or 150 ng/pL in 50 pL) were mixed with a buffer containing Tris-Acetate pH 8.0 73 mM, Potassium Acetate 60 mM, Magnesium Acetate 30 mM.
• the Exonuclease III was added either 20u in 2 pL buffer for 30 ng/pL specimen or 0,5 pL of stock solution 200u/pL for 150 ng/pL samples.
• the circularization method according to the invention provides high yields of circularized DNA molecules at increased DNA concentration, as indicated by the presence of two major bands: the circularized 4,528 bp and linear 1,901 bp both at expected molecular weight position.
• the linear 4,528 bp band and concatemers are not visible.
• the DNA molecules for transfection were circular 4,528 bp DNA molecules obtained with the use of the method according to the invention and the 6,711 bp plasmid DNA molecules, the parental DNA for production of circular 4,528 bp DNA molecules (see Example 10). Both DNA molecules contain a genetic construct for eukaryotic GFP gene expression.
• the cells were passaged into 24-well plate at 40 % to 70 % confluency in standard conditions.
• the transfection reagent JetOptimus® (Polyplus® transfection) was applied with four amounts of DNA 1,000 ng, 750 ng, 500 ng or 250 ng following the provided protocol. The cells were analyzed under the fluorescent microscope 24 hours after the cell transfection.
• Results Figure 16A-B

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